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UNIVERSITY OF HAWAI'! LIBRARY HIGH-THROUGHPUT TRANSIENT GENE EXPRESSION IN PLANT CELLS A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCES IN MOLECULAR BIOSCIENCES & BIOENGINEERING AUGUST 2005 By Leyang Huang Thesis Committee: Wei-Wen Su~ .Chariperson Monto Kumagai Yong-Soo Kim ACKNOWLEDGEMENTS I would like to express my gratitude to my advisor, Dr. Wei-Wen Su, for giving me the opportunity to work on this project. I appreciated his encouragement and guidance during my research and was impressed by his diligence and intelligence in these years. I would like to thank Dr. Monto Kumagai who gave me useful information and advice on molecular cloning techniques. Without his help, I could not complete my study. I am also grateful to Dr. Yong-Soo Kim for his review and valuable suggestions on my thesis. It was my pleasure to work with all my colleagues, including Alain, Aren Ewing, Bo Liu, Gabriel Peckham, Guocheng Du, Gwen, Ivo, Jennifer, Jose, Kaloian Nickolov, Madu, Malkeet Singh, Maribel, Peizhu Guan, Thara, and Yan Chen. I want to thank my parents, Chushen Huang and Yuying Xu, for their constant care and support. I am grateful to my relatives for their encouragement. I also enjoyed the friendship with my friends, especially Weij ing Wang, Hsiu-Ying Lin, Nan Zhang and Zijin Guo. This research was supported by the USDA TSTAR program award #200334135-13981. - 111 - ABSTRACT A variety of transient expression formats were modified from traditional gene transfer methods to improve the throughput of expression and characterization of gene products in cultured plant cells or protoplasts. The host cells were immobilized or confined in an array format such as well-less hydrogels and micro-wells. In the most promising system, genes of interests were delivered into plant protoplasts and cells via viral transfection and Agrobacterium-mediated transformation, respectively. The former (termed micro-transfection) is based on the polyethylene glycol (PEO)-mediated transfection of tobacco BY-2 protoplasts in a 96-well culture plate using recombinant tobacco mosaic virus (TMV) vectors; while the latter (termed micro-transformation) entails transformation of tobacco BY-2 cells in a 24-well format using Agrobacterium tumefaciens binary vectors. In micro-transfection studies, viral vectors encoding green fluorescent protein (OFP), red fluorescent protein (DsRed2) and rice a-amylase were constructed and used to demonstrate the utility of the system. Assay conditions were optimized based on the expression efficiency using OFP reporter gene. The transfection efficiency reached as high as 33% in 48 hours. In micro-transformation studies, transient OFP activities can be detectable after 3 or 4 days of co-cultivation with efficiency up to 10%. The precision and consistency of these systems were estimated by intra-assay and inter-assay coefficient of variations (CV). The transient expression systems developed in this research are attractive for highly parallel gene expression which could accelerate studies of gene functions, protein interactions and drug screening, at reduced scales and costs. - IV- TABLE OF CONTENTS ACKNOWLEDGEMENTS iii ABSTRACT iv LIST OF TABLES ix LIST OF FIGURES x CHAPTER 1. INTRODUCTION 1 1.1. Purpose of the study 1 1.2. Literature review 5 1.2.1. Green fluorescent protein (GFP) 6 1.2.2. Recombinant viral nucleic acid vectors 6 1.3. Research plan 8 1.4. References 9 CHAPTER 2. PROTOPLAST TRANSFECTION 2.1. Introduction 12 12 2.1.1. Protoplasts 12 2.1.2. Protoplast transformation 13 2.1.2.1. Electroporation 14 2.1.2.2. PEl-mediated transfection 15 2.1.2.3. PEG-mediated transfection 15 2.1.2.4. Other methods 15 2.1.3. 2.2. Transient expression system based on tobacco protoplasts Materials and Methods 16 16 2.2.1. Chemicals and reagents 16 2.2.2. Culture ofplant cells 17 2.2.3. Determination of cell viability 17 2.2.4. Examination of cell walls 18 2.2.5. Isolation of protoplasts 18 v 2.2.5.1. suspension cultured cells 18 2.2.5.2. Isolation ofmesophyll protoplasts from Nicotiana benthamiana 19 2.2.5.3. Preparation ofprotoplasts from tobacco BY-2 suspension cells 20 2.2.6. 2.3. Preparation of protoplasts from Nicotiana tobacum L. cv. Xanthi Transfection of protoplasts 20 2.2.6.1. Electroporation 20 2.2.6.2. PEl-mediated transfection 21 2.2.6.3. PEG-mediated transfection 21 Results and discussion 22 2.3.1. Optimization of protoplast isolation 22 2.3.2. Transient expression ofGFP in protoplasts 26 2.4. Concluding remarks 28 2.5. References 29 CHAPTER 3. WELL-LESS CELL ARRAY SySTEM 33 3.1. Introduction 33 3.2. Materials and Methods 38 3.2.1. Chemicals 38 3.2.2. Single layer hydrogel 39 3.2.3. Double layer hydroge1. 39 3.2.4. Hydrogelliquefication and Western blot of released viruses 40 3.3. Results and discussion 3.3.1. 41 PLL-coated single layer hydrogel.. 3.3.1.1. Optimization of protoplast attachment on hydrogels 41 41 3.3.1.1.1. Effect of PLL concentration 41 3.3.1.1.2. Effect ofPLL coating time 42 3.3.1.1.3. Effect of sodium alginate composition 42 3.3.1.1.4. Effect of sodium chloride washing 44 3.3.1.1.5. Effect of concanavalin A (Con A) 44 3.3.1.2. Gelliquefication and Western blot analysis of released virus VI 45 3.3.1.3. 3.3.2. Problems and modifications Double layer hydrogel. 46 46 3.3.2.1. Effect of polymer composition on virus release 47 3.3.2.2. Effect of pH on virus release 48 3.3.2.3. Problems and modifications 49 3.4. Concluding remarks 49 3.5. References 50 CHAPTER 4. MICRO-TRANSFECTION 52 4.1. Introduction 52 4.2. Materials and Methods 53 4.2.1. Materials 53 4.2.2. Preparation, inoculation and purification of viral vectors 53 4.2.2.1. In vitro transcriptions, encapsidation and inoculations 54 4.2.2.2. Dual inoculation ofNbenthamiana plants 55 4.2.2.3. Purification of virions 55 4.2.3. Micro-transfection of protoplasts 56 4.2.4. Western blotting 57 4.3. Results and discussion 4.3.1. 58 Adapt protoplast transfection in microtiter plates 58 4.3.1.1. Micro-transfection: TTOIA bGFP 60 4.3.1.2. Optimization of assay conditions 62 4.3.1.3. Western blot analysis of expressed proteins 64 4.3.2. Applications 65 4.3.2.1. Expression of red fluorescent protein (DsRed2) 65 4.3.2.2. Expression of rice a-amy,lase in micro-transfection system 69 4.3.2.3. Expression of multiple proteins 70 4.3.2.3.1. Dual inoculation 70 4.3.2.3.2. Multiple gene expression by a single vector. 71 4.3.2.3.2.1. Construction ofTTOSAI RFPKexGFP Vll 72 4.3.2.3.2.2. Expression of fusion fluorescent protein 75 4.4. Concluding remarks 77 4.5. References 78 CHAPTER 5. MICRO-TRANSFORMATION 5.1. Introduction 80 80 5.1.1. Agrobacterium-mediated transformation 80 5.1.2. Transient expression system based on tobacco cells 81 5.2. Materials and Methods 81 5.2.1. Chemicals 81 5.2.2. Agrobacterium binary vector 82 5.2.3. Agrobacterium-mediated transformation 82 5.2.3.1. Transformation ofBY-2 cells 82 5.2.3.2. Transformation ofBY-2 protoplasts 83 5.2.4. Western blotting 83 5.2.5. GUS activity assay 84 5.3. Results and discussion 84 5.3.1. Micro-transformation 84 5.3.2. Optimization of assay conditions 86 5.3.3. Assay quality control 88 5.4. Concluding remarks 89 5.5. References 90 CHAPTER 6. CONCLUSION AND RECOMMENDATION Vlll 92 LIST OF TABLES Table Page 2-1. Effect of enzyme composition on mesophyll protoplast isolation 23 2-2. Effect of enzyme composition on Xanthi protoplast isolation 23 2-3. Optimal conditions for tobacco protoplast isolation 26 3-1. Effect of PLL concentration on protoplast attachment. .42 3-2. Effect ofPLL coating time on protoplast attachment. .42 3-3. Effect of sodium alginate composition on protoplast attachment .43 3-4. Effect of Con A on protoplast attachment .45 6-1. microtiter plate-based transient gene expression systems 93 IX LIST OF FIGURES Figure Page 1-1. Organization of the TMV genome (Cann, 1997) 7 2-1. Effect of digestion temperature on Xanthi protoplast longevity 24 2-2. Effect of plating density on Xanthi protoplast culture 25 2-3. Fluorescence microscopy of freshly isolated BY-2 protoplasts 25 2-4. Fluorescent microscopy oftransfected BY-2 protoplasts expressing GFP 28 3-1. Chemical composition of alginate .34 3-2. Design of single-layer hydrogels 36 3-3. Double-layer hydrogel system .38 3-4. Western blot analysis of virions entrapped in single-layer hydrogels .45 3-5. Release ofTMV from hydrogels coated with different polymers .47 3-6. Effect of pH on the controlled release ofviruses .48 4-1. Virus transfection and replication 54 4-2. Flowchart of micro-transfection 59 4-3. N benthamiana plants expressing GFP in systemic leaves 61 4-4. Micro-transfection of tobacco BY-2 protoplasts with TTOIA bGFP 61 4-5. Effect of PEG concentration on micro-transfection 62 4-6. Effect of cell age on micro-transfection 63 4-7. Effect of PEG incubation time on micro-transfection 63 4-8. Time course of micro-transfection 64 4-9. Western blot analysis ofGFP expression using micro-transfection 65 - x- Figure Page 4-10. Viral vector encoding red fluorescent protein: TTOSAI DsRed2 67 4-11. N. benthamiana plants expressing RFP in systemic leaves 68 4-12. Micro-transfection of tobacco BY-2 protoplasts with TTOSAI DsRed2 69 4-13. a-amylase activity in transfected protoplasts 70 4-14. Co-transfection ofBY-2 protoplasts 71 4-15. Dual inoculation of N. benthamiana 71 4-16. Flowchart for vector construction: TTOSA 1 RFPKexGFP 74 4-17. Plasmid map ofTTOSAI RFPKexGFP 75 : 4-18. Infection ofTTOSAI RFPKexGFP in systemic leaves of N. benthamiana 76 4-19. Western blot analysis of extracts infected by TTOSAI RFPKexGFP 76 5-1. Agrobacterium-mediated transformation 85 5-2. Micro-transformation of BY-2 cells with Agrobacterium C58Cl m-gfp5-ER 85 5-3. Micro-transformation: Agrobacterium concentration 86 5-4. Time course of micro-transformation 87 5-5. Western blot analysis of micro-transformation 88 - XI- CHAPTER 1. INTRODUCTION 1.1. Purpose ofthe study Molecular biotechnology plays an important role in modem plant biology. The number of known genomes and genes are increasing rapidly. However, traditionally genes are studied one at a time, so that the throughput is very limited and the "whole picture" of gene function is hard to obtain. The completely annotated reference genomes of Arabidopsis thaliana and rice have been served as a starting point for the large-scale functional analysis of other plant genomes by comparative genomics. One strategy for the discovery of the presence of a trait and the function of unknown gene sequences in plants is to create a database of expressed sequence tags (ESTs) that can be used to identify expressed genes. This approach of randomly selecting and sequencing a large set of eDNA clones allows to put together a collection of sequence fragments of expressed genes. EST data may be used to identify gene products and thereby accelerate gene cloning. The most conclusive information about changes in gene expression levels can be gained from analysis of the varying qualitative and quantitative changes of messenger RNAs, proteins and metabolites (Holtorf et aI., 2002). New technologies have been developed to allow fast and highly parallel measurements of these constituents of the cell that make up gene activity. In recent years, DNA microarray has attracted tremendous interests among biologists (Schena et aI., 1995; Sanders and Manz, 2000). This technology allows massively parallel interrogation of gene expression on a single chip so that researchers can gain a better picture of the interactions among thousands of genes simultaneously. - 1- Cell-based arrays have become a fast and reproducible approach for therapeutic and diagnostic analysis in living cells or frozen cells. Stephan et al. (2002) developed a frozen cell array that allows for the analysis of a large number of cell types in a single experiment. This approach takes advantage of the cryopreservation of cells and provides a broad application base including antibody- and ligand-binding studies in a wellpreserved environment. The binding of antibody to a human glycoprotein was screened in 24 mammalian cell lines. Ziauddin and Sabatini (2001) developed a mammalian cell microarray using a technique called reverse transfection. In their gene expression system, mammalian cells were cultured on glass slides printed with sets of cDNA in expression vectors. After taking up the cDNA at each location, defined clusters of transfected cells with different plasmid DNAs were created and analyzed. In addition to the high-throughput characterization of gene functions, the cell microarray also has a potential as a method of screening for gene products involved in biological processes of pharmaceutical interest and as in situ protein microarrays for the development and assessment of leads in drug discovery (Bailey et aI., 2002). Because mammalian transfection methods generally do not work in plant cells, a fundamental change has to be made to create a plant cell mlcroarray. A highly efficient gene transfer system for plant cells is needed by plant scientists to achieve simultaneous transformation of a large number of genes for functional analysis of plant genes. It is most appropriate to study plant gene function in a plant cell environment because the potential alteration of protein functions due to differences in posttranslational processing would be minimized. Furthermore, functions of the expressed plant gene -2- could be identified by examining cellular phenotypes directly in the host. Kumagai et aI. (2002) have created genomic or cDNA libraries in recombinant viral nucleic acid vectors and used high-throughput robotics to facilitate the inoculation of Nicotiana benthamiana plants and functional genomic screening of a GTP binding protein. It usually takes one to two weeks to evaluate the expression in infected plants; while plant protoplasts are able to be transfected with viral vectors and express high level of recombinant proteins 24 hours post inoculation (Nagata et aI., 1981; Kikkawa et aI., 1982). Plant protoplasts are widely used in transient expression assays using electroporation or polyethylene glycol (PEG)-mediated transfection with foreign materials such as plasmid DNA, RNA or viruses (Dixon, 1994; Koop et aI., 1996). It has been shown that when protoplasts from a variety of plant systems were inoculated in vitro with TMV, virus infected and multiplied without causing necrosis in the protoplasts (Murakishi et aI., 1984). Thus the use of protoplast viral vector system should be amenable for highthroughput gene expression even though no report has been made. Agrobacterium tumefaciens binary vectors are also used as a tool for molecular biologists to introduce foreign genes into plant cells or protoplasts. Although Agrobacterium-mediated transformation is widely used to create transgenic plants in modem plant biology and agricultural biotechnology (An, 1985; Gelvin, 2003), it is also feasible to monitor transient expressions and analyze gene functions in a several days. The purpose of this study was to adapt these traditional gene transfer methods into transient expression systems that could be used to improve the throughput of gene expression and facilitate the characterization of gene products and the study of protein interactions. In one strategy, the concept of Ziauddin and Sabatini (2001) was adapted to -3- create a well-less plant cell based microarray based on the viral vector/protoplast system for high-throughput analysis of multiple gene products in parallel. We proposed to entrap recombinant viral vectors harboring distinct cDNAs in hydrogel spotted at defined locations on the surface of a glass slide: We hypothesized that cultured protoplasts anchored on the gel spotted areas may become transfected by the viruses released from the hydrogel, creating spots of cell clusters expressing defined cDNA with localized transfection. The plant cell microarray has several potential applications. It is useful as a high-throughput protein production platform for testing and optimizing foreign gene expression, expression of cDNA libraries, gene silencing experiments, creating protein chips, testing metabolic engineering strategies, screening of new herbicide targets, detecting protein-protein interactions, or for the discovery of gene products that alter cellular physiology, etc. The transfection of protoplasts or transformation of plant cells can be carried out in microtiter plates with a smaller scale. Through miniaturization, this pattern achieves economies of scale because only small quantities of potentially scarce biological samples or rare cell lines are necessary to assay large sets of genes (Bailey et aI., 2002). Furthermore, expressed gene products are accessible to a broader range of detection methods because current microtiter plate readers are typically able to detect signals, such as fluorescence or absorbance intensity, averaged over all cells in a well. The transient expression system developed in this research is composed of three components: hosts, gene delivery vectors and detection methods. The hosts were cultured plant cells or protoplasts immobilized or confined in an array format suitable for highthroughput applications. Gene delivery vectors contained recombinant tobacco mosaic - 4- VIruS (TMV), including purified Vlflons or in vitro viral RNA transcripts, and Agrobacterium tumefaciens binary vectors. The gene transfer in host cells was monitored using GFP as a reporter gene. Gene products were analyzed using methods such as fluorescent microscopy or spectroscopy, enzyme activity and Western Blot assay. The system is potentially used to produce individual residential or secreted proteins, coexpress multiple gene products and monitor expression level of interested genes using accompanying reporter genes. Therefore, it is attractive for highly parallel gene expression which could accelerate studies of gene functions, protein interactions and drug screening, at reduced scales and costs. 1.2. Literature review With the invention of recombinant DNA technology, it is possible to make a construct in vitro and then put it into a cell by transfection. Breakthrough techniques such as polymerase chain reaction (PCR), DNA sequencing, molecular cloning and DNA microarray have greatly accelerated the field of life sciences and created an enormous impact on plant molecular biology (Field, 2001). Complementary DNA (cDNA) encoding genes of interest are cloned into expression vectors and delivered into host cells by transfection or transformation methods. Because the transformation method using stable transgenic plants are time and resource consuming, the transient expression method is more suitable to analyze the gene functions in transfected cells because it is not only rapid but also free from the interference of chromatin structure. The transfer of genes in the dedicated host results in the expression of foreign proteins or changes in physiology of cells. Chimeric genes made by fusing a reporter gene for green fluorescent protein (GFP), red fluorescent protein (RFP) or p-glucuronidase (GUS) to the promoters or genes -5- of interest were constructed and used to monitor the efficiency of transcription and gene expression. The utility of different fluorescent proteins also facilitates the localization of gene products in living organisms or multiple labeling of tissues. 1.2.1. Green fluorescent protein (GFP) Green fluorescent protein (Prasher et aI., 1992; Roger, 1998) is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish Aequoria victoria. GFP is comprised of 238 amino acids and stable in neutral buffers up to 65°C, and displays a broad range of pH stability from 5.5 to 12. It fluoresces maximally when excited at 395 nm or 473 nm, and fluorescence emission peaks at 509nm. Unlike bioluminescent reporters, GFP requires no additional proteins, substrates or co-factors to emit light. When irradiated with UV light or blue light, it converted the blue emission of aequorin, a chemiluminescent protein, to green fluorescence, which enables the examination of gene expression and protein localization in situ and in vivo. In addition, the gene expression can be observed in real time. GFP has been expressed in bacteria, yeast, molds, plants, drosophila, zebrafish, and mammalian cells. GFP can function as a protein tag, as it tolerates Nand C-terminal fusion to a broad variety of proteins many of which have been shown to retain native function (Paramban et aI., 2004). Therefore GFP is one of the most convenient tools to analyze the efficiency of transient expression studied in this project. 1.2.2. Recombinant viral nucleic acid vectors Plant virus based vectors have been used to act as vehicles to deliver foreign genes into diverse plant hosts and express a variety of proteins in plants. Tobacco -6- mosaic virus (TMV) virions are 300-nm rod-shaped viral particles containing the viral genome and coat protein. The TMV genome consists of a 6.4-kb single stranded RNA comprising four open reading frames (ORPs) (Goelet et aI., 1982). During replication, these ORPs are transcribed and translated into the 126- and l83-kDa replicase proteins from the plus strand genomic RNA and the 30-kDa movement protein and 17.5-kDa coat protein from two subgenomic RNAs (Figure 1-1). The coat protein protects the virus from the environment and serves as a vehicle for transmission from one host cell to another. Figure 1-1. Organization of the TMV genome (Cann, 1997) 3' tRNAhis _ _ 3' 3' In a TMV based vector, the whole viral genome is cloned as a cDNA and inserted into a plasmid. Recombinant virions are then produced by transcription of the viral sequence on the plasmid into infectious RNA that is translated to produce proteins involved in replication, movement and encapsidation. One of the most successful transfection systems uses hybrid tobamoviruses to produce heterologous proteins in inoculated plants (Donson et aI. 1991, 1994; Kumagai et aI., 1993, 2000). -7- The eDNA encoding foreign proteins was subcloned into the 30-kDa protein coding region and placed under the control of the TMV-Ul coat protein subgenomic promoter. An additional sequence consisting of RNA subgenomic promoter from related tobamovirus and its coat protein sequence was also cloned to create the hybrid viral vector. By using two heterologous promoters to synthesize subgenomic RNAs for the foreign gene and coat protein gene respectively, the deletion of foreign inserts and loss of long distance viral movement due to the recombination between two repeated subgenomic promoter sequences was avoided. The resulting viral vectors are self-replicating, capable of systemic infection and stable transcription and expression of foreign genes in infected plants. 1.3. Research plan The goal of this project was to develop a transient gene expression platform amenable for high-throughput applications. To achieve this goal, there were three main tasks in this project. First, traditional gene transfer methods, including the transfection of protoplasts using viral vectors and the transformation of BY-2 cells with Agrobacterium binary vectors, were adapted into different formats and evaluated for their amenabilities to improve the throughput of transient expression. Second, the assay conditions were optimized based on the expression of GFP reporter gene. The precision and consistency of these systems were estimated by intra-assay and inter-assay coefficient of variations (CV). Third, the utility of the system to analyze gene function and potential protein interaction was demonstrated. Viral vectors encoding GFP, red fluorescent protein (RFP) and rice a-amylase were constructed and analyzed for gene expression. TMV-GFP and TMV-DsRed2 were also used to monitor the efficiency of co-transfection with multiple -8- constructs. Another viral vector encoding RFP and GFP joint by a Kex2p linker sequence (Jiang and Rogers, 1999) was constructed to demonstrate the multi-gene expression by one vector. These studies were carried out to explore the potential application of the transient expression system for protein interaction studies. 1.4. References An G., 1985. High efficiency transformation of cultured tobacco cells. Plant Physiology 79: 568-570. Bailey S.N., Wu RZ., Sabatini D.M., 2002. Application oftransfected cell microarrays in high-throughput drug discovery. Drug Discovery Today 7 (18): 1-6. Cann AJ., 1997. Principles of Molecular virology. Academic press, San Diego. p.74. Dixon RA, 1994. Application of protoplast technology. In: Dixon RA, Gonzales RA., Plant cell culture: a practical approach, 2nd ed. IRL press, Oxford. p.49. Donson J., Kearney C.M., HilfM.F., Dawson W.O., 1991. Systemic expression ofa bacterial gene by a tobacco mosaic virus-based vector. Proceedings ofthe National Academy of Sciences of USA 88: 7204-7208. Donson l, Dawson W.O., Granthan G.L., Turpen T.H., Turpen AM., Garger S.J., Grill L.K., 1994. Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes. US patent No. 5316931. Field S., 2001. The interplay of biology and technology. Proceedings of the National Academy of Sciences of USA 98 (18): 10051-10054. Gelvin S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiology and Molecular Biology Reviews 67 (1): 1637. Goelet P., LomonossoffG.P., Butler P.J.G., Akam M.E., Gait M.l, Karn J., 1982. Nucleotide sequence of tobacco mosaic virus RNA. Proceedings of the National Academy of Sciences of USA 79: 5818-5822. Holtorf H., Guitton M.C., Reski R, 2002. Plant functional genomics. Naturwissenschaften 89: 235-249. -9- Jiang L.W., Rogers J.C., 1999. Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells. The Plant Journal 18 (1): 23-32. Kikkawa R., Nagata T., Matsui C., Takebe I., 1982. Infection of protoplasts from tobacco suspension cultures by tobacco mosaic virus. Journal of General Virology 63: 451-456. Koop H.U., Steinmiiller K, Wagner H., RoBler C., Eibl C., Sacher L., 1996. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. P1anta 199: 193-201. Kumagai M.H., Turpen T.H., Weinzettl N., Della-Cioppa G., Turpen A.M., Donson J., . HilfM.E., Grantham G.L., Dawson W.O., Chow T.P., Piatak M., Grill L.K., 1993. Rapid high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector. Proceedings of the National Academy of Sciences of USA 90: 427-430. Kumagai M.H., Donson 1., Della-Cioppa G., Grill L.K, 2000. Rapid, high-level expression of glycosylated rice a-amylase in transfected plants by an RNA viral vector. Gene 245: 169-174. Kumagai M.H., Della-Cioppa G.R, Erwin R.L., McGee D.R, 2002. Method of compiling a functional gene profile in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation. US patent No. 6426185. Murakishi R., Lesney M., Carlson P., 1984. Protoplasts and plant viruses. Advances in Cell Culture 3: 1-55. Nagata T., Okada K, Takebe I., Matsui C., 1981. Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Molecular and General Genetics 184: 161-165. Paramban RI., Bugos RC., Su W.W., 2004. Engineering green fluorescent protein as a dual functional tag. Biotechnology and Bioengineering 86 (6): 687-697. Prasher D.C., Eckenrode V.K., Ward W.W., Prendergast F.G., Cormier M.J., 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111: 229-233. - 10- Roger Y.T., 1998. The green fluorescent protein. Annual Review of Biochemistry 67: 509-544. Sanders G.H.W., Manz A., 2000. Chip-based Microsystems for genomic and proteomic analysis. Trends in Analytical Chemistry 19 (6): 364-378. Schena M., Shalon D., Davis R.W., Brown P.O., 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467470. Stephan J.P., Schanz S., Wong A., Schow P., Wong W.L.T., 2002. Development ofa frozen cell array as a high-throughput approach for cell-based analysis. American Journal of Pathology 161 (3): 787-797. Ziauddin J., Sabatini D.M., 2001. Microarrays of cells expressing defined cDNAs. Nature 411: 107-110. - 11 - CHAPTER 2. PROTOPLAST TRANSFECTION 2.1. Introduction Plant protoplasts are widely used in transient expression studies because they are able to be transfected with foreign materials such as plasmid DNAs, RNAs or viruses and express high level of gene products 24 hours post inoculation (Nagata et aI., 1981; Kikkawa et aI., 1982). Thus the modification from protoplast transfection system is a promising strategy to improve the throughput of transient gene expression in a plant cell environment. Techniques related to the isolation, transfection and culture of protoplasts was explored and applied throughout this project. 2.1.1. Protoplasts Protoplasts are cell-wall-removed plant cells and are widely used as materials for cell fusion and transformation of foreign genes (Dixon, 1994). Researches with protoplasts are carried out in fields of plant sciences such as plant cell genetics, plant pathology, photosynthesis, cell division, and cell-wall biosynthesis, etc. Protoplasts have been successfully isolated, cultured and manipulated from different tissues including leaves, shoots, embryos and seedlings, suspension cultured cells and callus, of a wide range of plant species. The plant cell walls were commonly degraded by a combination of enzymes including cellulase, pectinase and hemicellulase. To remove undigested tissues, cellular and subcellular debris, protoplasts were purified by different methods including: (1) flotation on dense sucrose solution (Blackhall et aI., 1994) or Percoll solution (Somerville et aI., 1981), (2) density gradient centrifugation (Masuda et aI., 1989), (3) repeated centrifugation - 12 - and resuspension (Nagata et aI., 1981), and (4) aqueous two phase system separation (Kanai and Edwards, 1973). Isolated protoplasts are usually cultured and regenerated In liquid media or embedded in semi-solid or solid media containing agar, agarose or alginate. To maintain the osmotic potential of the protoplasts, sugars or sugar alcohols such as sucrose, mannitol and sorbitol were added into buffers and media used for protoplast isolation and culture. The viabilities of protoplasts are evaluated using vital stains such as fluorescein diacetate (FDA), Evan's blue, phenosafranin (Franklin and Dixon, 1994) and 2,3,5triphnyl tetrazolium chloride (TTC) (Watanabe et aI., 1992). In general, the concentration of a protoplast solution is measured by hemacytometer and adjusted to the appropriate cell density for growth. 2.1.2. Protoplast transformation Transformation is the process of introduction of foreign genes into cells. New genes and therefore new traits are introduced into a cell or organism. The successful protoplast transformation depends on the efficiency of transferring DNA into protoplasts and protoplast purification. After removal of the cell walls, isolated protoplasts are capable of uptaking foreign materials, such as DNA or plasmids (Koop et aI., 1996), RNA (Goodall et aI., 1990), liposomes (Watanabe et aI., 1982), viruses (Kikkawa et aI., 1982; Takebe, 1984), bacteria (Marton, 1984) and proteins (Wu et aI., 2003). The cell membranes can be made permeable by physical treatments such as microinjection, particle bombardment and electroporation or chemical methods mediated by polyethylene - 13 - glycol (PEG), poly-L-omithine (PLO), poly-L-Iysine (PLL) or polyethyleneimine (PEl). During transformation, the plasma membranes were penetrated without permanently damaging the cells, allowing the entry of foreign gene delivery vectors. After cultured in solid media with approximate osmotic pressure, transfected protoplasts were able to divide and regenerated into transgenic plants. In protoplast fusion, hybrid cells were generated from protoplasts isolated from different plants speCIes. Because protoplasts were uniform and could be infected simultaneously with a number of plant viruses, protoplasts were used extensively in studies of virus infection, replication, cytopathology, protection and genetic engineering of virusresistant plants (Murakishi et aI., 1984). Infection by plant viruses is initiated by virus particles entering host cells. Recombinant virions assembled in protoplasts can be extracted and detected using various methods such as immunodetection and infectivity assay (Maule et aI., 1980). 2.1.2.1. Electroporation Electroporation has been widely used to introduce foreign DNA, viral RNA and viruses into bacteria, plant protoplasts (Watanabe et aI., 1987; Mas and Beachy, 1998; Valat et aI., 2000) or plasmolyzed plant cells (Wu and Feng, 1999; Kosciaiiska and Wypijewski, 2001). It is a simple, effective and non-toxic technique to transfect plant cells. Under an electric field, temporary pores or channels are generated on the lipid bilayers of cell membranes and stimulated the uptake of exogenous vectors. The formation of such pores and channels is reversible and, therefore, cells are able to recover and express foreign genes. - 14- 2.1.2.2. PEl-mediated transfection Polyethylenimine (PEl) is a positively charged polymer that can form complexes with DNA, RNA or virions and thus facilitate the gene delivery into living cells (Kikkawa et aI., 1982; Boussif et aI., 1995; Godbey et aI., 1999). Under most conditions where transfections take place, viruses and protoplast surfaces are negatively charged. The presence of PEl promotes the formation of virus-polycation complex which can approach the protoplast membrane more easily (Wood, 1985). Other polycations that mediate and achieve the transfection of protoplasts include poly-L-arginine, poly-L-Iysine (PLL) and poly-L-ornithine (PLO) (Maule et aI., 1980; Takebe, 1984). 2.1.2.3. PEG-mediated transfection Polyethylene Glycol (PEG) is able to alter the properties of the protoplast membrane and is efficient in promoting membrane fusion and the infection of plant protoplasts by viral nucleoprotein, nucleic acid and RNA-encapsulated liposomes. PEG treatment of protoplasts is a useful, simple and cost-efficient alternative to electroporation (Koop et aI., 1996). 2.1.2.4. Other methods Transfection of protoplasts can potentially be achieved using other novel gene transfer techniques including magnetofection and peptide-mediated delivery. Magnetofection has been proposed as a simple and highly efficient method for gene therapy applications (Scherer et aI., 2002; Krotz et aI., 2003). This technology utilizes magnetic fields to enhance and target the delivery of gene vectors that form complexes with magnetic nanoparticles, toward the host cells. - 15 - Wu et al (2003) demonstrated that proteins could be delivered into plant protoplasts via a peptide-mediated method by using the Chariot reagent (Active Motif, Carlsbad, CA) which forms non-covalent complex with the macromolecules to be delivered such as proteins, peptides and antibodies. 2.1.3. Transient expression system based on tobacco protoplasts Tobacco protoplasts were chosen as the main host in this project to develop the high-throughput transient gene expression systems. Parameters including source of protoplasts, enzyme composition, digestion time, and purification methods were studied to improve the quality and viability of isolated protop1asts. Recombinant tobacco mosaic virus (TMV) vectors (Kumagai et aI., 1993) encoding modified green fluorescent protein (m-gfp5-ER, Haseloff et aI., 1997) were used as a model system to study the transfer of foreign genes into protoplasts. Different methods, including electroporation, PEl and PEG mediated transfection, were compared based on the transfection efficiency and the adaptability for high-throughput applications. 2.2. Materials and Methods 2.2.1. Chemicals and reagents Murashige and Skoog (MS) basal medium salt mixtures were obtained from Phyto Technology Laboratories (Shawnee Mission, KS). Cellulase "onozuka" RS was purchased from Yakult Pharmaceutical Ind. Co. Ltd. (Tokyo, Japan). Pectolyase Y-23 was purchased from Kyowa Chemical Products Co. Ltd. (Osaka, Japan). Cellulase R10 and Driselase were distributed by Karlan Research Products Corporation (Santa Rosa, CA). Cellulysin, Macerase, fluorescein diacetate (FDA) and Calcofluor White M2R were obtained from Sigma (St. Louis, MO). Linear PEl (MW 25 kD) and - 16- branched PEl (MW 10 kD and 70 kD) were purchased from Polysciences Inc. (Warrington, PA). 2.2.2. Culture of plant cells The suspension culture of tobacco cells (Nicotiana tobacum L. cv. Xanthi) was maintained in MS basal medium supplemented with Gamborg vitamins, 20 giL sucrose, 1 mgIL 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L kinetin, pH being adjusted to 5.6. The cells were cultured at room temperature in a reciprocating shaker at 110 r.p.m. and subcultured weekly at 15-20% inoculums in fresh medium. Tobacco Nicotiana tobacum L. cv Bright Yellow 2 (BY-2) cells were kindly provided by Dr. Ken Matsuoka ofRIKEN in Japan. Suspension culture of BY-2 cells were maintained in MSD medium containing 4.3 giL MS salt, 0.2 mg/l 2,4-D, 1 mg/L thiamine-Hel, 0.2 gIL KH2P04, 0.1 gIL myo-inositol and 30 gIL sucrose. The pH of the medium was adjusted with KOH to a range between 5.6 and 5.8. The cells were subcultured weekly by transferring 2 ml of the cell suspension into 100 ml of fresh MSDmedium. 2.2.3. Determination of cell viability The viability of cells and protoplasts were determined by fluorescein diacetate (FDA). FDA (Larkin, 1976) is a non-fluorescent molecule that is able to pass through the cell membrane and enter cells freely. The detection of the stain is dependent upon the ability of esterases in viable cells to cleave FDA and release fluorescein, which is retained in the cytoplasm. Stock solution of FDA (5 mglml in acetone) was diluted with culture medium to a working concentration of 0.02%. Equal volumes of the working solution and cells were mixed to give a final FDA concentration of 0.01 % - 17 - and examined using an Olympus B~60 fluorescence microscope under blue light illumination. Protoplasts which are viable emit a green/yellow fluorescence, while non-viable protoplasts don't fluoresce. Cells were counted using a Fuchs-Rosenthal hemacytometer with a field depth of 0.2 mm. The viability of cells was estimated as the percentage of the number of living fluorescent cells out of the total number of cells in the same field. At least 200 protoplasts were counted for each determination. 2.2.4. Examination of cell walls The efficiency of cell wall removal was assessed using a fluorescent brightener called Calcofluor White (Nagata and Takebe, 1970; Hahne et aI., 1983). The fluorescent stain binds to ~-linked glucosides such as cellulose or chitin within the cell wall and is useful for visualization of cell wall during cell wall biosynthesis (Galbraith, 1981). 5 Jil of 0.1 % (w/v) of Calcofluor White M2R in wash buffer containing 0.4 M mannitol, 10 mM CaCh and 5 mM MES (pH5.5) were added to 100 Jil of protoplast solution and stained at room temperature for 5 minutes. After rinsed three times to remove excess dye, protoplasts were examined using a fluorescent microscope with Omega filter set XF136 (365WB50/400DCLP/450AF58). The existing cell walls produced an intense blue fluorescence when examined under UV illumination. 2.2.5. Isolation of protoplasts 2.2.5.1. Preparation of protoplasts from Nicotiana tobacum L. cv. Xanthi suspension cultured cells One gram fresh weight of tobacco N tobacum L. cv. Xanthi suspension cells were harvested four days after subculture by centrifuging at 1000 g for 1 minute - 18 - and resuspended in 10 ml of CPW13M solution containing 5mM 4morpholinoethanesulfonic acid (MES), 27.2 mg KH2P04, 101 mg KN03, 1480 mg CaCh2H20, 246 mg MgS04"7H20, 0.025 mg CuS04"5H20, 0.16 mg KI and 130 g mannitol (pH5.8) for 3 hours. Plasmolyzed cells were resuspended in 10 ml of 0.2 /lm filter-sterilized enzyme solution consisting of 1% cellulase RS, 0.1 % pectolyase Y-23, dissolved in CPW13M solution, pH 5.7. The cell/enzyme mixture was incubated at 50 rpm on a gyratory shaker at 30°C for 40 minutes. Freshly isolated protoplasts were filtered through 41 /lm nylon cloth and centrifuged at 100 g for 3 minutes using a swinging bucket rotor to remove the enzyme solution, followed by washing in CPW13M solution twice. Protoplasts were then cultured in CPW solution supplemented with 0.2 mg/12, 4-D, 0.2 mg/l kinetin and 0.3M sucrose, pH 5.8 at a cell density of2x105/ml in Petri dishes at room temperature. 2.2.5.2. Isolation of mesophyll protoplasts from Nicotiana benthamiana Leaves from one-month-old N. benthamiana plants were surface sterilized in 20% bleach for 15 minutes. After rinsed in sterile ddH20 three times, the leaves were blot dried on filter paper in laminar flow hood. One gram fresh weight of leave tissues was cut into thin strips using sterile blades and transferred to 25 ml of 0.2 /lm filter-sterilized enzyme solution consisting of 2% Cellulysin and 0.5% Macerase dissolved in CPW13M solution at pH 5.7. The plate was incubated at 40 rpm. on a gyratory shaker at 30°C for 3 hours. Enzyme solutions containing protoplasts were filtered through 41 /lm nylon cloth and centrifuged at 100 g for 3 minutes to remove undigested tissue and debris. After washed in CPW13M solution - 19- three times, protoplasts were resuspended in CPW13M supplemented with 0.2 mg/l 5 2,4-D and 1% of sucrose (pH 5.8) at a density of2x10 /ml at room temperature. 2.2.5.3. Preparation of protoplasts from tobacco BY-2 suspension cells Protoplasts were isolated under aseptic conditions from four-day-old suspension culture of Nicotiana tobacum L. cv. BY-2 cells. One gram fresh weight ofBY-2 cells was collected by centrifuging at 1000 g for 1 minute using a swinging bucket rotor in a bench-top centrifuge (Eppendorf 5702R) and resuspended in 10 ml of 0.2 J.1m filter-sterilized enzyme solution consisting of 1% Cellulase RS and 0.1 % Pectolyase Y-23 dissolved in CPW wash buffer containing 0.4 M mannitol, 10 mM CaCh and 5 mM MES (pH5.6). Incubation for 45 to 50 minutes at 40 rpm on a gyratory shaker at 30°C was sufficient to convert majority of the cells into protoplasts. The protoplast solution was centrifuged at 200 g for 1 minute to remove the enzymes. After additional washes with CPW wash buffer. Protoplasts were then cultured in protoplast culture medium (PCM) containing 4.3 gil MS salt, 0.2 mg/l 2,4-D, 1 mg/l thiamine-HCL, 0.37 gil KHZP04, 0.1 gil myo-inositol, 10 gil sucrose 5 and OAM mannitol (pH 5.8) at a cell density of2x10 /ml at room temperature. 2.2.6. Transfection of protoplasts 2.2.6.1. Electroporation 2x 106 freshly isolated BY-2 protoplasts were resuspended in 0.8 ml of ice-cold electroporation buffer consisting of 0.4 M mannitol, 70 mM KCI and 5 mM MES (pH5.7). 1 J.1g of purified recombinant TMV carrying the modified GFP reporter gene was added and gently mixed with protoplasts. The mixture was incubated on - 20- ice for 5 minutes and transferred into a pre-chilled electroporation cuvette with a distance of 0.4 cm between the electrodes. Electroporation was performed with the BTX Electro Cell Manipulator 600 units (BTX, San Diego) at 200 V and 320 J.lF. The electroporated sample was incubated on ice for 30 minutes and then diluted into 10 ml of protoplast culture medium (PCM). Transient expression of GFP was monitored using an Olympus BX60 fluorescent microscope with U-M41Ol7 endow GFP filter (HQ470/40, Q495lp, HQ525/50, Chroma Technology Corp.) after 48 hours. The transfection efficiency was estimated as the ratio of the number of transfected protoplasts to the total number of viable protoplasts. The viability of protoplasts was estimated by FDA. Each sample was counted for three times and averaged. At least 100 protoplasts were counted for each determination. 2.2.6.2. PEl-mediated transfection 1 J.lg of purified TMV-GFP virions were resuspended in 2 ml of 0.02 M potassium citrate buffer (pH5.2) containing 0.4 M mannitol and 1.6 J.lg/ml PEl. After incubated at 25°C for 10 minutes, the virus/PEI complex was added to 2 ml of protoplast solution (4x 105 cells/ml) and kept at 25°C for another 10 minutes. Protoplasts were washed three times in CPW wash buffer and cultured in PCM at a density of 2x 105/ml up to 48 hours. Protoplasts expressing GFP was visualized under a fluorescence microscope equipped with a Macrofire digital camera. 2.2.6.3. PEG-mediated transfection Approximately 2x 106 protoplasts were resuspended in a minimum volume of 0.4 M mannitol CPW wash buffer and mixed with 1 J.lg of recombinant TMV - 21 - virions harbored with the GFP cassette. 500 III of 40% PEG 6000 solution was then added dropwise to the mixture and incubated for 1 minute. Protoplasts were then diluted with 5 ml of wash buffer and kept at room temperature for 10 to 20 minutes, followed by three washes to remove excess PEG and viruses. The repeating wash step was carried out by resuspending protoplasts in wash buffer and centrifuging at 200 g for 1 minute. Protoplasts were cultured in PCM at a density of 2x 105/ml and analyzed for transient expression in 48 hours. Green fluorescent protoplasts were visualized under a fluorescence microscope and the transfection efficiency was estimated as described in 2.2.6.1. 2.3. Results and discussion 2.3.1. Optimization of protoplast isolation Potoplasts were isolated from leaves of Nicotiana benthamiana and suspension cultured cells of Nicotiana tobacum L. cv. Xanthi and Nicotiana tobacum L. cv. Bright Yellow-2 (BY-2). Although the enzymatic isolation of protoplasts is a wellestablished technique, the conditions have to be optimized for different species and tissues. Different factors affecting the quality and viability of protoplasts have been studied, including enzyme composition, digestion temperature, digestion time, plating density, purification methods, and growth phase of source materials. The plant cell wall is mainly composed of cellulose, hemicellulose and pectin and requires a mixture of enzymes to degrade it effectively. Different combinations of wall-degrading enzymes were tested for optimal yield and viability of protoplasts isolated from N benthamiana leaves (Table 2-1) and N tobacum Xanthi suspension cells (Table 2-2). The protocols of protoplast isolation were described in section 2.2.5. - 22- Table 2-1. Effect of enzyme composition on mesophyll protoplast isolation 6 Cellulysin (%) Macerase (%) Yield (10 protoplasts/g FW) Viability (%) 1.0 0.25 0.132 50.0 1.0 0.5 2.103 54.0 1.0 0.75 1.398 55.0 1.5 0.25 0.242 50.0 1.5 0.5 2.383 57.9 1.5 0.75 1.070 38.4 2.0 0.25 1.160 75.0 2.0 0.5 7.944 77.5 2.0 0.75 4.029 58.9 Table 2-2. Effect of enzyme composition on Xanthi protoplast isolation Enzyme composition Viability time course Yield (hours) (protoplasts/ml) 1% Cellulase RS, 0.1 % Pectolyase Y23 2% Cellulase RIO, 1% Driselase, 0.1 % Pecto1yase Y23 1% Cellulase RS, 1% Driselase, 0.1 % Pectolyase Y23 0 18 42 66 5x105 90% 80% 75% 60% 2x10 5 88% 75% 66% 50% 2x10 5 50% 33% 10% 0 4 60% 40% 25% 10% 1% Cellu1ysin, 1% Driselase, 0.1 % 5x10 Macerase Note: All enzymes were dissolved in CPW13M wash solution. The isolation of protoplasts from N tobacum Xanthi cell suspension was further optimized based on the viability and longevity of protoplast culture. Suspension cells growing at exponential phase, usually four days after subculture, provide an active metabolizing protoplast culture. A plasmolysis treatment, where cells were incubated - 23 - in CPW13M solution for 3 hours, was found to be beneficial to protoplast isolation because cytoplasm was detached from the cell wall under high osmotic pressure before digestion. Most plasmolyzed cells were converted to protoplasts in 40 minutes. More protoplasts with better longevity were released when digestion was carried out in Petri dishes at 30°C where the enzyme activities were optimal (Figure 2-1). Figure 2-1. Effect of digestion temperature on Xanthi protoplast longevity. Protoplasts were plasmolyzed for 3 hours and then incubated with enzyme solution containing 1% cellulase RS and 0.1 % Pectolyase Y23 for 40 minutes at 30°C and room temperature, respectively. After digestion, protoplasts were cultured at a cell density of 2x 105 cells/ml in liquid media at room temperature. Different approaches including membrane filtration, aqueous two-phase separation and Percoll gradient were examined for Xanthi protoplast purification. Due to the low yield, these methods did not result in significant improvement in purity. Filtration was regarded as the simplest method to get rid of undigested cell clumps. Broken protoplasts or cell debris were removed mostly by repeated centrifugation and resuspension. After purification, protoplasts cultured at a cell density of 2x 105 cells/ml in liquid media were found to have a better longevity (Figure 2-2). - 24- Figure 2-2. Effect of plating density on protoplast culture 80% -r--------------------...., l: 70% .8= 60% ~... 50% ~ 40% = C 30% == ,J:J 20% :>= 10% 0% -+-----""--"'''"""""''"-O+----.................- ' ' ' " I - - - ' =..........L....--+--.....=.;;,:.I....---I Plating density (protoplasts/ml) Note: Protoplasts were incubated with I% cellulase RS and 0.1 % Pectolyase Y23 at 30°C. After filtered through nylon cloth and washed with CPW13M solution, protoplasts were cultured at different cell density at room temperature. The optimal conditions for the isolation and purification of all three types of tobacco protoplasts are summarized in Table 2-3. Compared to the other sources (i.e. N tobacum Xanthi cell suspension and N. benthamiana leaves), BY-2 cells have a higher growth rate and are more homogeneous, and using BY-2 cells, protoplasts could be produced with much higher yield and viability (Figure 2-3). Therefore, BY-2 protoplasts were used as the main host cells in subsequent experiments throughout this project to develop high-throughput transient expression systems. Figure 2-3. Fluorescence microscopy of freshly isolated protoplasts from tobacco N tobacum BY-2 suspension-cultured cells. Protoplasts were stained with FDA and visualized using Chromas U-M41017 EN GFP filter set. - 25 - Table 2-3. Optimal conditions for tobacco protoplast isolation Species Tissue Enzyme composition Enzyme volume Nicotiana tobacum Nicotiana Nicotiana tobacum L. cv. L. cv. Xanthi benthamiana BY-2 suspension cells leaves suspension cells 1% cellulase RS, 2% Cellulysin, 0.1% pectolyase Y- 0.5% Macerase, 23, pH 5.7 pH 5.7 10 ml/g fresh weight 25 ml/gFW CPW salts, 5 mM mannitol, pH 5.8 CPW13M CPW salts, 0.2 mgll culture 2, 4-D, 0.2 mgll medium kinetin,O.3M (PCM) sucrose, pH 5.8 10 ml/g fresh weight (FW) 10 mM CaCh, 5 mM MES, pH 5.6 (CPW13M) Protoplast Pectolyase Y-23, pH 5.6 CPW salts,OA M mannitol, MES, 130 gil Wash buffer 1% Cellulase RS, 0.1 % 4.3 gil MS salt, 0.2 mgll 2,4CPW13M, 0.2 D, 1 mgll thiamine-HCL, mgll 2, 4-D, 10 gil 0.37 gil KH2P04, 0.1 gil sucrose, pH 5.8 myo-inositol, 10 gil sucrose, OAM mannitol, pH 5.8 Digestion 40 minutes (2-3 time hours plasmolysis) Purification filtration, filtration, methods centrifugation centrifugation 3~5 5~1O 3 hours 45-50 minutes centrifugation Yield (lOt> protoplasts 20~30 IgFW) Note: Enzymes are dissolved in corresponding wash buffers. FW-fresh weight. 2.3.2. Transient expression of GFP in protoplasts The first step to develop a protoplast-based transient expression system was to select a highly efficient and effective transfection method. Different approaches, - 26- including PEG, PEl or PLO mediated transfection and electroporation were tested. Transient gene expressions were achieved using virions from Nicotiana benthamiana infected with a recombinant TMV vector that carried modified GFP (m-gfp5-ER, Haseloff et aI., 1997) as a reporter gene. The transfection efficiencies of various approaches were compared and some adjustments were made so that the selected method could be applied in the high-throughput transient expression system. Electroporation is a widely used method to transfer target genes into cultured cells or protoplasts (Register, 1994). Although electroporation was a simple and effective technique and was able to transfect up to 20~25% of protoplasts, it is more difficult and costly to implement electroporation in a high-throughput format. In addition to having a concentrated virus solution with high infectivity and a freshly prepared suspension of active protoplasts, viral infection is facilitated by bringing both viruses and protoplasts into a close proximity using polycations such as poly-L-ornithine (PLO) or polyethyleneimine (PEl). BY-2 protoplasts were transfected with different types of PEl, including the ExGen 500 in vitro transfection reagent, branched PEl with a molecular weight of 10 k:D and 70 k:D and linear PEl (MW 25k:D), respectively. The resulting transfection efficiency was up to 1O~ 15%, which is lower than the following PEG-mediated method. The infection of protoplasts by viral RNA or particles could also be mediated by high concentration of PEG. After mixing with concentrated PEG solution, BY-2 protoplasts and viruses were washed with buffer containing calcium and cultured in PCM up to 48 hours. Transient expression of GFP could be observed in infected protoplasts under blue light excitation with a transfection efficiency of up to 33% - 27- (Figure 2-4). Among the transfection methods tested in this study, PEG-mediated transfection was the most effective and was subsequently adapted for high-throughput expression as to be described in the following chapters. Figure 2-4. Fluorescent microscopy of BY-2 protoplasts transfected with recombinant TMV virions harboring GFP reporter gene. The transfection was mediated by PEG and protoplasts were observed in 48 hours. (a) lO-fold, (b) 40-fold magnification. 2.4. Concluding remarks The isolation and purification of tobacco protoplasts from various materials were optimized and summarized in Table 2-3. Protoplasts with high yield and viability were obtained from suspension-cultured BY-2 cells more easily and reproducibly. The resulting active metabolizing protoplasts were able to be transfected by recombinant TMV and were used as the main hosts in subsequent experiments throughout this project to develop high-throughput transient expression systems. Among different gene delivery approaches, PEG-mediated transfection was most efficient and effective when using GFP as a reporter gene. Different modifications were applied to improve the throughput of the transient expression system afterwards. - 28- 2.5. References Blackhall N.W., Davey M.R., Power J.B., 1994. Isolation, culture and regeneration of protoplasts. In: Dixon R.A., Gonzales R.A., Plant cell culture: a practical approach, 2nd ed. IRL press, Oxford. p.27. BoussifO., Lezoualc'h F., Zanta M.A., Mergny M.D., Scherman D., Demeneix B., Behr J.P., 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine. Proceedings of the National Academy of Sciences of USA 92: 7297-7301. Dixon R.A., 1994. Application of protoplast technology. In: Dixon R.A., Gonzales R.A., Plant cell culture: a practical approach, 2nd ed. IRL press, Oxford. p.49. Franklin C.I., Dixon R.A., 1994. Initiation and maintenance of callus and cell suspension cultures. In: Dixon R.A., Gonzales R.A., Plant cell culture: a practical approach, 2nd ed. IRL press, Oxford. p.1. Galbraith D.W., 1981. Microfluorimetric quantitation of cellulose biosynthesis by plant protoplasts using Calcofluor White. Physiologia Plantarum 53: 111-116. Godbey W.T., Wu K.K., Mikos A.G., 1999. Polyethylenimine and its role in gene delivery. Journal of Controlled Release 60: 149-160. Goodall GJ., Wiebauer K., Filipowicz W., 1990. Analysis ofpre-mRNA processing in transfected plant protoplasts. Methods in Enzymology 181: 148-161. Hahne G., Herth W., Hoffmann F., 1983. Wall formation and cell division in fluorescence-labelled plant protoplasts. Protoplasma 115: 217-221. Haseloff J., Siemering, K.R., Prasher D.C., Hodge S., 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the National Academy of Sciences of USA 94: 2122-2127. Kanai R., Edwards G.E., 1973. Purification of enzymatically isolated mesophyll protoplasts from C3,C4, and crassulacean acid metabolism plants using an aqueous dextran-polyethylene glycol two-phase system. Plant Physiology 52: 484-490. - 29- Kikkawa H., Nagata T., Matsui c., Takebe I., 1982. Infection of protoplasts from tobacco suspension cultures by tobacco mosaic virus. Journal of General Virology 63: 451-456. Koop H.D., Steinmiiller K., Wagner H., RoBler C., Eibl C., Sacher L., 1996. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199: 193-201. Koscianska E., Wypijewski K., 2001. Electroporated intact BY-2 tobacco culture cells as a model of transient expression study. Acta Biochimica Polonica 48 (3): 657-661. Krotz F., Wit C., Sohn H.Y., Zahler S., Gloe T., Pohl D., Plank C., 2003. Magnetofection-a highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo. Molecular Therapy 7 (5): 700-710. Kumagai M.H., Turpen T.R., Weinzettl N., Della-Cioppa G., Turpen A.M., Donson J., HilfM.E., Grantham G.L., Dawson W.O., Chow T.P., Piatak M., Grill L.K., 1993. Rapid high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector. Proceedings of the National Academy of Sciences ofDSA 90: 427-430. Larkin P.J., 1976. Purification and viability determinations of plant protoplasts. Planta 128: 213-216. Marton L., 1984. Transformation of tobacco cells by coculture with Agrobacterium tumefaciens. Cell Culture and Somatic Cell Genetics of Plants 1: 514-521. Mas P., Beachy R.N., 1998. Distribution ofTMV movement protein in single living protoplasts immobilized in agarose. The Plant Journal 15 (6): 835-842. Masuda K., Kudo-Shiratori A., Inoue M., 1989. Callus formation and plant regeneration from rice protoplasts purified by gradient centrifugation. Plant Science 62: 237246. Maule AJ., Boulton M.I., Wood K.R., 1980. An improved method for the infection of cucumber leafprotoplasts with cucumber mosaic virus. Phytopath. Z. 97: 118-126. Murakishi H., Lesney M., Carlson P., 1984. Protoplasts and plant viruses. Advances in Cell Culture 3: 1-55. - 30- Nagata T., Takebe I., 1970. Cell wall regeneration and cell division in isolated tobacco mesophyll protop1asts. P1anta 92: 301-308. Nagata T., Okada K., Takebe I., Matsui c., 1981. Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Molecular and General Genetics 184: 161-165. Register J.C.III, 1994. Studies with viruses. In: Dixon RA., Gonzales RA., Plant cell culture: a practical approach, 2nd ed. IRL press, Oxford. p.56. Scherer F., Anton M., Schillinger D., Henke J., Bergemann C., KrUger A., Gansbacher B., Plank C., 2002. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy 9: 102-109. Somerville C.R, Somerville S.C., Ogren W.L., 1981. Isolation of photosynthetically active protoplasts and chloroplasts from Arabidopsis thaliana. Plant Science Letters 21: 89-96. Takebe I., 1984. Inoculation of protoplasts with plant viruses. Cell Culture and Somatic Cell Genetics of Plants 1: 492-502. Valat L., Toutain S., Courtois N., Gaire G., Decout E., Pinck L., Mauro M.C., Burrus M., 2000. GFLV replication in e1ectroporated grapevine protoplasts. Plant Science 155: 203-212. Watanabe M., Watanabe Y., Shimada N., 1992. Colorimetric estimation ofleafprotoplast potential-to-divide by use of2,3,5-triphenyl tetrazolium chloride. J. Plant Physiology 141: 111-114. Watanabe Y., 0000 T., Okada Y., 1982. Virus multiplication in tobacco protoplasts inoculated with tobacco mosaic virus RNA encapsulated in large unilamellar vesicle 1iposomes. Virology 120: 478-480. Watanabe Y., Meshi T., Okada Y., 1987. Infection of tobacco protoplasts with in vitro transcribed tobacco mosaic virus RNA using an improved electroporation method. FEB Letters 219 (1): 65-69. Wood K.R, 1985. Tissue culture methods in phytopathology. I-Viruses. In: Dixon RA., Plant cell culture: a practical approach. IRL press, Oxford. p.193. - 31 - Wu F.S., Feng T.Y., 1999. Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells. Plant Cell Reports 18: 381-386. Wu Y., Wood M.D., Tao Y, Katagiri, 2003. Direct delivery of bacterial avirulence proteins into resistant Arabidopsis protoplasts leads to hypersensitive cell death. The Plant Journal 33: 131-137. - 32- CHAPTER 3. WELL-LESS CELL ARRAY SYSTEM 3.1. Introduction Cell-based arrays have become an attractive approach for therapeutic and diagnostic analysis to facilitate gene expression and characterization of gene products in a highly parallel pattern. Ziauddin and Sabatini (2001) developed a mammalian cell microarray using a technique called reverse transfection. In their gene expression system, mammalian cells were cultured on glass slides printed with sets of cDNA in expression vectors. After taking up the cDNA at each location, defined clusters of transfected cells with different plasmid DNAs were created and analyzed. Because mammalian transfection methods generally do not work in plant cells, a fundamental change has to be made to create a plant cell microarray. The isolation, purification and viral transfection of plant protoplasts were discussed in the previous chapter. In this chapter, the concept of Ziauddin and Sabatini (2001) was adopted to create a well-less plant cell microarray based on the viral vector/protoplast system for high-throughput analysis of multiple gene products in parallel. We proposed to entrap recombinant viral vectors harboring distinct cDNAs in hydrogel spotted at defined locations on the surface of a glass slide. We hypothesized that cultured protoplasts anchored on the gel spotted areas may become transfected by the viruses released from the hydrogel, creating spots of cell clusters expressing defined cDNA with localized transfection. One unique requirement in the well-less transfection system is the ability to control the release of viral vectors at will. In this study we proposed to use an alginate-polycation - 33 - microcapsule system for that purpose. Hydrogel entrapment is a useful cell immobilization technique. Among the various hydrogels, alginate is one of the most commonly used. Typical alginate polycation system comprises two components: a core material of alginate hydrogel and a polyanion-polycation complex membrane. Alginates (Yang and Wright, 1998) are anionic polysaccharides extracted from brown seaweeds, such as Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria hyperborean, and are composed of 1, 4-linked ~-D-mannuronic (M) and a-L-guluronic (G) acid residues with regions of alternating blocks (Figure 3-1). In the presence of divalent cations such as calcium or barium, these residues are crosslinked to form gel cores for microcapsules. Figure 3-1. Chemical composition M of alginate. a, The monomers of G a alginate, Haworth conformation. M, A-D-mannuronate· fJ , G, a-L. guluronate. b, The alginate chain, G G M M G b chain conformation. c, Symbolic representation of the MMMMGMGGGGGMGMGGGGGGGGMMGMGMGGM alginate chain (Thu et aI., 1996). G·bIOCk There are two kinds of gelation techniques, external and internal gelation (Dulieu et aI., 1998). The traditional procedure of dropping alginate solution into a calcium chloride buffer is known as external gelation because alginate residues are crosslinked by external calcium ions diffused into the droplet. The resulting hydrogel beads are inhomogeneous - 34- with higher alginate concentration on the surface, leading to more binding of polycation. More homogeneous beads are obtained by internal gelation, where the pH of alginate solutions containing fine, insoluble calcium carbonate microcrystals are reduced from 7.5 to 6.5. The internal calcium source solubilizes and triggered the gelation of alginate. There are different types of alginate (Serp et aI., 2000) varying in molecular weights and guluronic acid to mannuronic acid ratio (GIM ratio). Guluronic acid residues have higher charge density than mannuronic acid residues and are responsible for the mechanical strength of the capsule (Thu et aI., 1996). The main functions of the core materials are to entrap cells rapidly under mild conditions and to serve as a template for binding of polycation. Poly-L-Iysine (PLL) is a polyamine composed oflysine monomeric amino acids and is widely used to coat solid substrates like glass slides. When adsorbed to the culture surface, PLL increases the number of positively charged sites available for cell binding. When alginate beads are suspended in a PLL solution, the positively charged PLL amino groups are able to interact with the negatively charged carboxyl groups on alginate residues and form a thin membrane network near the bead surfaces. Chitosan is a polyglucosamine polysaccharide derived from the partially deacetylation of chitin, a major component in crustacean shells (Kim et aI., 1998). It is positively charged due to the amino groups on its glucose backbones and able to form complex membrane with alginate via ionic interaction. Chitosan is a biocompatible polyelectrolyte and processes a wide range of useful properties in various applications including cosmetics, water treatment, biodegradable materials, and microcapsule implants for controlled release in drug delivery. - 35 - Alginate polycation microcapsules (Thu et aI., 1996) were commonly used as the materials for implantable bioartificial pancreas in medical treatments of diabetes and liver diseases. Semipermeable microcapsules (Goosen et aI., 1985) were formed by encapsulating biologically active islets or cells in spherical calcium alginate beads, which were coated with PLL. The semipermeable membrane served as a diffusion barrier to protect the islets from immune rejection of islet transplantation (De Vos et aI., 1993). Long-term in vivo biocompatible and durable microcapsules were thus developed and found to be biologically effective. In the presence of chelators such as phosphate, citrate, lactate or EDTA, calcium cations in the alginate network are displaced, leading to the liquefication of capsules. The polycation membrane can improve the strength of the beads and helps to form liquid-core capsules or so-called hydrogels when the gel cores are dissolved in phosphate or citrate buffers (Chang et aI., 1996; Quong and Neufeld, 1998). In a study in this chapter, alginate polycation hydrogel spots were arrayed onto glass slides to create a well-less cell array. Recombinant TMV vectors were mixed and entrapped in the biocompatible alginate gels coated with PLL or chitosan. Upon gel liquefication by citrate or phosphate buffer, released viruses were diffused into the surrounding environment and interacted with attached or immobilized plant protoplasts. Based on this concept, two cell array systems were designed and evaluated for transient gene expression. A PLL-coated single-layer alginate system was first developed (Figure 3-2). Sodium alginate solutions containing recombinant viral vectors were arrayed on the surface of solid supports like glass slides. The slides were immediately immersed into a CaCh - 36 - solution. A hydrogel created by the crosslink between alginate and calcium ions was then coated with PLL to form a thin polycation membrane on the surface of the anionic calcium alginate gel via electrostatic interactions. Slides deposited with arrays of minute hydrogel spots that contain recombinant viral vectors were then incubated in plant protoplasts solution. Negatively charged protoplasts were attached onto the polycation membrane of the hydrogel via ionic interaction. Protoplasts grown on the gel surface were expected to become transfected by the viruses or viral transcripts released from the hydrogel. Spots of cell clusters with localized transfection, surrounded by a field of nontransfected protoplasts, were thus created and analyzed. The efficiency of protoplast attachment on PLL coated gel spots and the effects of entrapment and release of viruses were explored. Figure 3-2. Design of single-layer hydrogels Alginite e:::t o Incuballan WI Protoplast Suspenslan " II '------' ~--"'::c... Protoplast Attachment . .. I . GID c=l -- ..... .' Nan-transfec:t.d pratopillsts Tranlfect8d prmopllsts ". e Liquefied Alginite Polymer Membrane wi Modulated Permelbllity - 37 - A double-layer hydrogel system was modified from the single-layer system (Figure 3-3). Protoplasts have been reported to remain viable and metabolically active in calcium alginate beads, which act as solid supports for plant regeneration from embedded protoplasts (Larkin et aI., 1988; Dovzhenko et aI., 1998). In addition to the alginate layer containing viral vectors, an outer layer entrapping protoplasts was formed and covered by another polycation membrane. Thus the design became a "sandwich" format comprised of two hydrogel layer confined by two membranes. Different polycation membranes were used to obtain the controlled release of the entrapped viral vectors. The inner membrane was designed to be permeable for virus after gelliquefication in phosphate buffer (PBS), while the outer membrane should have poor permeability so that released virus were restricted in outer hydrogels and interacted with protoplasts. Glass slides with arrayed hydrogels were then incubated in protoplast culture media for transfection to take place. Factors affecting the permeability of polycation membrane and the resulting controlled release of entrapped viruses were studied in the double layer hydrogel system. Figure 3-3. Double-layer hydrogel system Protoplasts in alginate Transfected protoplasts LiquefiC,ti; pH 7.0 ,.....L.~-_----l...:...L., ,~:'f:ti':"1J!.~ I . . Virus in alginate 3.2. Materials and Methods 3.2.1. Chemicals Sodium alginate (low viscosity and medium viscosity), chitosan, poly-L-Iysine solution (high MW), poly-L-Iysine hydrochloride (PLL) with molecular weight - 38- between 15,000 and 30,000 (low MW), 30,000 and 70,000 Daltons (medium MW), Nitro blue tetrazolium (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) were obtained from Sigma (St. Louis, MO). 3.2.2. Single layer hydrogel The surfaces of glass slides were coated with 0.005% PLL solution for ten minutes and dried at room temperature. Recombinant TMV virions purified from infected tobacco Nicotiana benthamiana plants were diluted 1000 fold in a 1.5% sodium alginate solution (low viscosity). A microarray was created by spotting 2~1 each of the viral alginate mixture onto the glass slides, followed by immersing the slides into a 1.5% CaCh solution for 5 minutes. After washed in saline (0.9% NaCI), the calcium alginate gel spots were coated with 0.02% PLL solution for 30 minutes. The Ca-alginate-PLL hydrogels was then washed and stored in water for the subsequent experiments. Leak-free entrapment of virions was examined by Western blot analysis. The isolation, viability assay and culture of protoplasts from tobacco (Nicotiana tobacum L. cv. Xanthi) cell suspensions were carried out as described in Chapter 2. Single-layer hydrogel microarrays were incubated with protoplast solution for 30 minutes and washed with water twice. Protoplasts attached onto the gel spots were examined under a microscope and the extent of adhesion was estimated. 3.2.3. Double layer hydrogel In the double layer format, the inner gel layer was created according to the above method, except that the calcium alginate gel spots were coated with 0.15% chitosan dissolved in 0.02M sodium acetate buffer (pH 5.0) for 1 hour. Freshly isolated - 39- tobacco protoplasts were suspended in a sodium alginate solution containing 0.4 M mannitol. The mixture was deposited onto the first layer of hydrogel that was coated with chitosan and crosslinked in a 1.5% CaCh solution for 10 minutes. The two-layer hydrogel spots were washed with water and then immersed into a 0.02% PLL solution for 30 minutes. After washed with water, the double layer microarrays were incubated in protoplast culture medium for the subsequent experiments. 3.2.4. Hydrogelliquefication and Western blot of released viruses PLL or chitosan-coated sodium alginate hydrogel spots containing recombinant TMV virions were created in microwells of a 96-well plate using methods described in 3.2.2. Hydrogels were liquefied in a 50 III of 50 mM sodium citrate buffer for 5 minutes and then incubated in 50 III of water. Aqueous samples were taken from each well and suspended in sample loading buffer containing 50 mM Tris-HCI (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.25% p-mercaptoethanol and 0.0025% Bromophenol blue. Proteins were then separated by a 12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). In the double layer system, after liquefied in a sodium citrate buffer, hydrogels were incubated in 50 III of 0.2 M sodium phosphate buffer, pH 7.0 for 30 minutes before taking samples. Western blot analysis of released viruses was carried out by electro-transferring separated proteins onto a polyvinylidene fluoride (PVDF) microporous membrane (Millipore). The membrane were blocked with 5% milk dissolved in TBST for 1 hour, followed by another one-hour-incubation in a 1:2000 rabbit anti-TMV VI coat antibody solution. After three 5-minute washes in TBST, the membrane was - 40- incubated in a secondary antibody solution containing 1:2000 alkaline phosphatase (AP) conjugated goat-anti-rabbit IgG for 45 minutes. Antibody bound coat proteins were detected using NET and BCIP as substrates for AP. 3.3. Results and discussion 3.3.1. PLL-coated single layer hydrogel 3.3.1.1. Optimization of protoplast attachment on hydrogels A PLL-coated single-layer alginate system was first developed. Parameters including PLL concentration, coating time, alginate type and concentration, additives like NaCI and lectins were optimized to enhance the attachment of protoplasts to the surface of the hydrogels. 3.3.1.1.1. Effect of PLL concentration The first step to develop the cell array was to coat the glass slide with PLL solution before arraying the gel spots. Since gel spots were unable to stay firmly on the slide surface after immersed into CaCh solution, ionic interaction between PLL and negatively charged sodium alginate was necessary for the restriction and formation of the hydrogels. 0.005% PLL solution was used to coat the slides and was found to be sufficient to facilitate the array of gel spots. Isolated plant protoplasts were able to attach onto glass slides or 1.5% hydrogels pre-coated with PLL solution. The effect of PLL concentration on protoplast attachment was examined in Table 3-1. Less than 0.02% of PLL coating was not effective on attachment because the thickness of alginate-PLL complex membrane decreased at lower PLL concentration (Vandenbossche et aI., 1993). The adhesion of PLL was less specific because not only protoplasts, but - 41 - also cell debris in protoplast solution were able to attach onto the gel spots. As PLL concentration increased, more cell debris was found to be attached. Thus 0.02% ofPLL solution was used to coat the hydrogels in future experiments. Table 3-1. Effect ofPLL concentration on protoplast attachment PLL concentration 0.005% 0.01% 0.02% 0.05% 0.1% Protoplasts attached - + ++ ++ ++ Debris attached - - - + + Note: --less than 10 per spot, +-10 to 50 per spot, ++-50 to 100 per spot 3.3.1.1.2. Effect of PLL coating time 1.5% calcium alginate gel spots were incubated in a 0.02% PLL solution for 10,30 and 60 minutes, respectively (Table 3-2). According to De Vos et aL (1993), an increased exposure time of microcapsules to PLL solution allowed an enhanced binding of PLL, which attached more protoplasts. However, as the coating time increased up to one hour, more cell debris was also observed on the hydrogel surfaces. An incubation time of 30 minutes was sufficient to form a PLL-alginate complex membrane for protoplasts adhesion. Table 3-2. Effect ofPLL coating time on protoplast attachment Note: Coating time (minutes) 10 30 60 Protoplasts attached ++ +++ +++ Debris attached + ++ +++ +-10~50 per spot, ++-50~100 per spot, +++-more than 100 per spot 3.3.1.1.3. Effect of sodium alginate composition Sodium alginate with low and medium viscosity was used to form hydrogels, respectively. Various concentration of sodium alginate solution - 42- including 1.0%, 1.5% and 2.0% was examined for effects on protoplast adhesion. For medium viscosity sodium alginate, only 1.0% and 1.5% was tested because the 2.0% solution was very viscous and difficult to handle. Binding rate, the total amount bound and the stability of the PLL membrane are related to the composition of the alginate. From Table 3-3, the extent of attachment was relatively higher when sodium alginate solutions with low viscosity were used. It was probably because the diffusion of PLL into the alginate gel network was faster in low viscosity hydrogels, leading to a thicker PLL membrane with more positive charged sites for protoplast attachment. Binding of PLL to alginate gel was mainly governed by the amount of dissociable negative charges on the bead surface (Thu et aI., 1996). When higher concentration of sodium alginate was used, there were more negative charges on the surface of hydrogels, which interacted with PLL and decreased available positive charges for protoplast attachment. Thu et ai. also concluded that the stability of hydrogels was enhanced as the alginate concentration in the core increased. Considering the effects of both charge density and gel strength, 1.5% of sodium alginate solution with low viscosity was used in the following experiments. Table 3-3. Effect of sodium alginate composition on protoplast attachment Sodium alginate viscosity low Sodium alginate concentration medium 1.0% 1.5% 2.0% 1.0% 1.5% Protoplasts without washing + ++ ++ + + attached washed with saline ++ +++ ++ ++ + Note: +-1O~50 per spot, ++-50~100 per spot, +++-more than 100 per spot - 43 - 3.3.1.1.4. Effect of sodium chloride washing A washing step with saline solution (0.9% NaCI) was applied on hydrogels before PLL coating. During the wash excess calcium cations on the gel surface were removed, generating more available negative charges for the binding of PLL. In Table 3-3, more protoplasts were observed to be adhered on gel spots produced after saline washing treatment. 3.3.1.1.5. Effect of concanavalin A (Con A) We observed that more protoplasts were anchored onto PLL coated glass slides than PLL coated hydrogels (data not shown). It is likely that a portion of the positive charges on PLL membrane were neutralized by the negatively charged alginate gels and thus the available positive charges were reduced. To improve the attachment of protoplasts, another coating of Concanavalin A (Con A) was applied onto the PLL-coated hydrogels. Concanavalin A (Con A) is a type of plant lectins, which can selectively bind to carbohydrates of cell surfaces and agglutinate various cells including protoplasts (Singh et aI., 1999). Con A reacts with the ring form of non- reducing a-D-glucose and a-D-mannose. At pH 7.0 it forms a tetramer with four sugar binding sites and has optimal activity to interact with cells. Con A was commonly used in adhesion and aggregation of cells, cell recognition and drug targeting studies (Glimerlius et aI., 1978; Bornman et aI., 1987). 250 J,lg/ml of Con A was prepared in 0.05M Tris-HCI buffer solution (pH7) containing 0.15M KCI and O.OIM CaCho When Con A was coated directly on alginate gel surfaces, few protoplasts were attached. The resulting gel spots were - 44- dissolved in sodium citrate buffer, indicating that Con A was not sufficient to act as the only membrane for cell arrays. An additional coating of Con A was thus used on PLL-coated slides to enhance the attachment of protoplasts. From Table 3-4, although Con A increased the amount of attached protoplasts by about 30%, but the extent of attachment was still not sufficient to cover the whole spot even highly concentrated protoplast solutions was dropped onto the hydrogels. Table 3-4. Effect of Con A on protoplast attachment Hydrogel Protoplast attached (per spot) Sodium citrate treatment Alginate-Con A <10 Dissolved in 5 min Alginate-PLL 100-120 Not dissolved Alginate-PLL-Con A about 150 Not dissolved 3.3.1.2. Gelliquefication and Western blot analysis of released virus The entrapment and release of recombinant virions was confirmed by Western blot analysis using primary antibodies against TMV coat proteins, which had a molecular weight of about 17.5 kDa (Figure 3-4). M 2 4 5 6 30kD- 1 kn- Figure 3-4. Western blot analysis 0 virions entrapped in single-layer hydrogels. M-Rainbow molecular weight marker, 1-3-Diluted TMV standard, 4-TMV in sodium alginate solution, 5-Hydrogel containing TMV in water, 6-Released TMV in sodium citrate buffer. - 45- The interaction between PLL and alginate formed a strong complex which stabilized and strengthened the ionic gel network. The permeability was thus reduced and controlled so that leak-free entrapment of recombinant virions could be achieved when the gel was exposed to water (Figure 3-4, lane 5). The core of hydrogels could be liquefied by exposing the gel to an aqueous citrate of phosphate buffer. Viral vectors released from the liquid-core hydrogels were able to be detected by Western blot and a TMV-coat-protein specific antibody (Figure 3-4, lane 6). 3.3.1.3. Problems and modifications Although the single layer hydrogel system was simple and amenable to highthroughput analysis theoretically, there were some obstacles in this approach. First, the extent of protoplast attachment was not sufficient to cover the entire gel spots, even if an additional coating of Con A seemed to enhance the attachment. Second, when using as a cell array, viral vectors carrying different genes would diffuse into media and caused interference by interacting with protoplasts located on other spots in the array. Third, the viral transfection efficiency was low due to the poor longevity or viability of attached protoplasts. 3.3.2. Double layer hydrogel To resolve the problems associated with the single-layer plant cell arrays, a double-layer hydrogel system was developed. By varying factors including the composition of the polycation membrane, polymer molecular weight and pH of the phosphate buffer, the porosity of the membrane can be modulated to optimize the controlled release of viral vectors. - 46- 3.3.2.1. Effect of polymer composition on virus release Hydrogels entrapping viral vectors were coated with four polymers: (1) chitosan, (2) low molecular weight (MW) PLL, (3) medium MW PLL, (4) high MW PLL. After liquefied in sodium citrate buffer, released viruses were detected by Western blot analysis against virus coat protein. From Figure 3-5, chitosan was more permeable to virus than PLL with lower molecular weight and was selected as the inner membrane. According to Gaserod et aI. (1998), PLL has a larger charge density than chitosan due to a shorter monomer length, leading to stronger electrostatic interaction and increased binding. Thus the porosity was smaller in PLL membrane and fewer viruses were released. The effect of PLL MW on membrane permeability is attributed to the extent of polycation penetration into the alginate network and its interactions with the hydrogel matrix (Machluf et aI., 1997). The smaller the MW of PLL, the more it can penetrate into the alginate matrix and, therefore, formed a thicker membrane. Less virus was released from PLL membrane with lower MW, as observed in Figure 3-5. PL PM PH Figure 3-5. Release ofTMV from hydrogels coated with different polymers. C-ehitosan PL-PLL (MW 15-30kD) PM-PLL (MW 30-70kD) PH-PLL (MW 70-150kD) - 47- 3.3.2.2. Effect of pH on virus release Hydrogels containing recombinant virus were coated with chitosan and low molecular weight PLL solution, respectively. After liquefication liquid-core gel spots were incubated in 0.2M sodium phosphate buffer (PBS) with pH 5.8, 6.5, 7.0 and 8.0. Optimal amounts of virus were released from chitosan membrane when the pH of PBS was 7.0, while fewer viruses were diffused out of low MW PLL !Figure 3-6, lane 5). At pH 6.0 or higher, the polymer chain in chitosan was shrinked due to electrostatic repulsion, leading to an increased membrane pore size and higher permeability of membrane (Okhamafe et aI., 1996). M 2 3 4 5 6 M 2 4 6 o o Figure 3-6. Effect of pH on the release of virus from chitosan inner membrane (left) and PLL (low molecular weight) outer membrane (right). M-Rainbow molecular weight marker, I-Diluted TMV standard, 2-Hydrogel containing TMV in H 20, 3-6-Hydroge1 in 0.2M sodium phosphate buffer, pH 5.8, 6.5, 7.0, 8.0. Therefore, the optimized double layer cell array system applied two layers of hydrogels confmed by PLL (low MW) outer membrane and chitosan inner membrane. Liquefication of gel cores and controlled release of viral vectors were carried out in 0.2M sodium phosphate buffer, pH 7.0. - 48- 3.3.2.3. Problems and modifications The double layer hydrogel design was a delicate cell array system suitable for the combination of different hosts and gene delivery vectors entrapped in separate hydrogels. Upon liquefication and pH treatment, viral vectors in inner alginate gels were diffused through the inner membrane and interacted with host cells in the outer hydrogel layer. An outer membrane with poor permeability was used to restrict the leakage of viruses. However, the transfection efficiency of protoplasts based on GFP reporter was extremely low. Apparently transfection could not be achieved by simple incubation of protoplasts and viruses. Although PEG greatly facilitate the uptake of viral vectors during transfection, the diffusion of PEG in alginate was not efficient to achieve high level of transient expression. Furthermore, high inhibitory activity of alginate against tobacco mosaic virus infection was reported by Sano (1999). Alginate caused TMV particle to form large raft-like aggregates and thus reduced the infectivity. 3.4. Concluding remarks The well-less cell array system utilized the cell entrapment or immobilization methods, creating alginate gel spots coated with a layer of polycation membrane, to confine viral vectors or protoplasts in a microarray format. Although this system was novel, simple and amenable for high-throughput transient expression, the efficiency of transfection was not sufficient for the detection of expressed genes as discussed previously. An efficient transfection system utilizing commercial multiwell culture plates was developed and described in the following chapter. - 49- 3.5. References Bornman C.H., Zachrisson A., 1987. Immobilization of plant protoplasts using microcarriers. Methods in Enzymology 135: [37] 421-433. Chang H.N., Seong G.H., Yoo I.K, Park J.K, Seo J.H., 1996. Microencasu1ation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules. Biotechnology and Bioengineering 51: 157-162. De Vos P., Wolters G.H.J., Fritschy W.M., Van Schilfgaarde R, 1993. Obstacles in the application of microencapsulation in islet transplantation. The International Journal of Artificial Organs 16 (4): 205-212. Dovzhenko A., Bergen D., Koop H.D., 1998. Thin-alginate-layer technique for protoplast culture of tobacco leaf protop1asts: shoot formation in less than two weeks. Protoplastma 204: 114-118. Dulieu C., Poncelet D., Neufeld RJ., 1998. Encapsulation and immobilization techniques. In: Kuhtreiber W.M., Lanza RP., Chick W.L., Cell Encapsulation Technology and Therapeutics. Birkhauser, Boston. p.3. Gasemd 0., Smidsmd 0., Skjak-Brrek G., 1998. Microcapsules of alginate-chitosan I: A quantitative study ofthe interaction between alginate and chitosan. Biomaterials 19: 1815-1825. Glime1ius K, Wallin A., Eriksson T., 1978. Concanavalin A improves the polyethylene glycol method for fusing plant protop1asts. Physiologia Plantarum 44: 92-96. Goosen M.F.A., O'Shea F.M., Gharapetian H.M., Chou S., Sun A.M., 1985. Optimization of microencapsulation parameters: semipermeable microcapsules as a bioartificial pancreas. Biotechnology and Bioengineering 27: 146-150. Kim S.K, Choi lH., Balmaceda E.A., Rha C.K, 1998. Chitosan. In: Kuhtreiber W.M., Lanza RP., Chick W.L., Cell Encapsulation Technology and Therapeutics. Birkhauser, Boston. p.15!. Larkin P.J., Davies P.A., Tanner G.J., 1988. Nurse culture oflow numbers of Medicago and Nicotiana protoplasts using calcium alginate beads. Plant Science 58: 203210. - 50- MachlufM., Regev 0., Peled Y., Kost J., Cohen S., 1997. Characterization of microencapsulated liposome systems for the controlled delivery of liposomeassociated macromolecules. Journal of Controlled Release 43 (1): 35-45. Okhamafe A.O., Amsden B., Chu W., Goosen M.F.A., 1996. Modulation of protein release from chitosan-alginate microcapsules using the pH-sensitive polymer hydroxypropyl methylcellulose acetate succinate. Journal of Microencapsulation 13 (5): 497-508. Quong D., Neufeld RJ., 1998. DNA protection from extracapsular nucleases, within chitosan- or poly-L-Iysine-coated alginate beads. Biotechnology and Bioengineering 60 (1): 124-134. Sano Y., 1999. Antiviral activity of alginate against infection by tobacco mosaic virus. Carbohydrate Polymers 38: 183-186. Serp D., Cantana E., Heinzen C., Von Stockar D., Marison I. W., 2000. Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization. Biotechnology and Bioengineering 70 (1): 41-53. Singh RS., Tiwary A.K., Kennedy J.F., 1999. Lectins: sources, activities, and applications. Critical Reviews in Biotechnology 19 (2): 145-178. Thu B., Bruheim P., Espevik T., Smidsmd 0., Soon-Shiong P., Skjak-Brrek G., 1996. Alginate polycation microcapsules. I. Interaction between alginate and polycation. Biomaterials 17: 1031-1040. Vandenbossche G.M.R, Van Oostveldt P., Demeester J., Remon J.P., 1993. The molecular cut-off of microcapsules is determined by the reaction between alginate and polylysine. Biotechnology and Bioengineering 42: 381-386. Yang H., Wright J.R, 1998. Calcium alginate. In: Kuhtreiber W.M., Lanza RP., Chick W.L., Cell Encapsulation Technology and Therapeutics. Birkhauser, Boston. p.79. Ziauddin J., Sabatini D.M., 2001. Microarrays of cells expressing defined cDNAs. Nature 411: 107-110. - 51 - CHAPTER 4. MICRO-TRANSFECTION 4.1. Introduction Although the well-less cell array system explored in Chapter 3 was novel and amenable for high-throughput analysis, protoplast transfection was not achieved because of the limitation of system described previously. Instead of entrapping protoplasts and viruses in alginate gel spots, another efficient transient expression format, which incorporated commercial microtiter plates to create the boundary, was developed in this chapter. This system was termed micro-transfection and was modified from the polyethylene glycol (PEG)-mediated transfection (Koop et ai. 1996) of plant protoplasts with recombinant viral vectors. Compared to regular transfection where a large amount of protoplasts and viruses were used, micro-transfection used a small volume of protoplasts and viruses to analyze transient expression in commercial 96-well plates. Although not reported, the protocol for micro-transfection had to be modified in order to achieve an efficient gene transfer in a scale which was less than 1% of the regular transfection. Through miniaturization, this pattern achieves economies of scale because only small quantities of potentially scarce biological samples or rare cell lines are necessary to assay large sets of genes (Bailey et aI., 2002). Furthermore, expressed gene products are accessible to a broader range of detection methods because current microtiter plate readers are typically able to detect signals, such as fluorescence or absorbance intensity, averaged over all cells in a well. Recombinant viral vectors encoding green fluorescent protein (GFP), red fluorescent protein (RFP) and rice a-amylase were used to demonstrate the application of micro- 52 - transfection. Assay conditions were optimized based on the transient GFP expression level in protoplasts. As a prerequisite for studying protein interaction using viral vectors, the strategies to express multiple proteins in plants or plant protoplasts were explored. In one strategy, both TMV-GFP and TMV-DsRed2 were inoculated into the hosts and the efficiency of co-transfection was monitored. In another strategy, viral vector encoding RFP and GFP connected by the Kex2 protease recognition sequence (Jiang and Rogers, 1999) was constructed as a model to express multiple proteins using a single vector. 4.2. Materials and Methods 4.2.1. Materials Restriction and cloning enzymes were obtained from New England Biolabs (Beverly, MA). Falcon 96-well tissue culture plates were purchased from Fisher Scientific Co. (Houston, TX). The mMessage mMachine high yield capped RNA transcription SP6 kit was obtained from Ambion (Cat #1340, Austin, TX). 4.2.2. Preparation, inoculation and purification of viral vectors The process of virus transfection and replication is described in Figure 4-1. After the construction of viral plasmid DNA encoding the TMV open reading frames (ORFs) and foreign gene, the viral vector was linearized and converted into infectious in vitro RNA transcripts by SP6 DNA-dependent RNA polymerase. The viral RNAs were then mechanically inoculated onto the lower leaves of Nicotiana benthamiana plants. About one to two weeks after inoculation, TMV were assembled and spread in the whole plant, causing host symptoms and producing recombinant proteins in mesophyll cells or interstitial fluid. Recombinant virions were purified from the - 53 - systemic leaves located at the 1/3 top of infected plants and used to transfect tobacco BY-2 protoplasts via micro-transfection. Figure 4-1. Virus transfection and replication Viral plasmid DNA Restriction enzyme digestion (KpnI) In vitro transcription reaction ---''"--- __ Mechanical inoculation onto N. benthamiana . . . . L . . _ ~ Recombinant Virus .---. Virus purification PEG-mediated Transfection (BY-2) ---''"-- 4.2.2.1. In vitro transcriptions, encapsidation and inoculations In hybrid viral vectors, the open reading frames (ORF) for target genes were placed under the control of tobamovirus coat protein subgenomic promoter. The viral plasmid was digested with KpnI and used as the linear template for in vitro transcription reaction using SP6 DNA-dependent RNA polymerase. Synthesized RNA was diluted in FES solution (7.508 gil glycine, 10.452 gil K2HP0 4 , 10 gil - 54- sodium pyrophosphate, 10 gil bentonite and 10 gil celite) and mechanically inoculated onto the lower leaves of 3 to 4 week-old N. benthamiana. For the encapsidation of viral RNA, 10 J.lI of the in vitro transcripts were incubated with I mg/ml of coat protein solution in 100mM sodium phosphate buffer, pH7.0 overnight at room temperature. Nicotiana benthamiana plants were mechanically inoculated with the encapsidated virus particles on the next day. When using GFP as a reporter gene, the transient GFP activities could be visualized under blue light excitation after 6 days. 4.2.2.2. Dual inoculation of N.benthamiana plants A mixture (1 J.lI each) of recombinant tobamovirus TTOIA bGFP and TTOSAI DsRed2 encoding GFP and RFP, respectively, was diluted in FES solution and inoculated onto the lower leaves of N.benthamiana plants. In another experiment, the two virions were inoculated separately onto different leaves of N.benthamiana. Expression of fluorescent proteins was analyzed by examining infected leaf discs under a fluorescent microscope using filter set for GFP and RFP. 4.2.2.3. Purification of virions Recombinant virions were isolated from infected leaves of N. benthamiana plants one to two weeks after inoculation using a method modified from Gooding and Herbert (1967). Two grams of leaves were ground in liquid nitrogen and suspended in 50 ml of 0.5M Na2HP04-KH2P04 buffer (pH 7.2) containing 1% f3- mercaptoethanol. The crude extract was filtered through cheesecloth, followed by adding 240 J.lI of n-butanol and vortexing every few minutes over a 15 minute - 55 - period. Leaf materials were removed by centrifuging the extract at 13200 rpm in an Eppendorf tabletop centrifuge for 30 minutes and filtering the supernatant through cheesecloth. The filtrate was mixed with 1/10 volume of 40% PEG solution and centrifuged at 13200 rpm for 15 minutes. The pellet was resuspended in 0.5 ml of 10 mM phosphate buffer, pH 7.2 and centrifuged at 10000 rpm for 10 minutes. 1/10 volume of 7M NaCl and 40% PEG solutions were added to the supernatant respectively and incubated for 15 minutes. After centrifuged at 12000 rpm for 15 minutes, the TMV pellets were resuspended in 200 !J.l of 10 mM phosphate buffer, pH 7.2. The concentration of purified virions was determined by UV spectrophotometer. In general, the OD260 of a 1mg/ml TMV solution equals to 2.7 when measured in a cuvette with 1 cm path-length. 4.2.3. Micro-transfection of protoplasts Protoplasts were isolated from 4-day-old suspension cultured tobacco BY-2 cells as described in Chapter 2 and were resuspended in 0.4 M mannitol CPW wash buffer at a density of 107 cells/m!. In each well of a Falcon 96-well culture plate, 8 !J.l of the protoplast solution were mixed with 2 !J.l of recombinant TMV vectors carrying the target gene cassette. 70 !J.l of 40% PEG 6000 solution were added to the mixture and incubated for 1 minute without shaking (actual PEG concentration was 35%). Protoplasts were then diluted with 160 !J.l of wash buffer and kept at room temperature for 15 minutes. About 3/4 volumes of the liquid in individual wells were removed using a handmade pipette tip connected to a vacuum chamber. Protoplasts were resuspended in 160 !J.l of wash buffer and sit for 15 minutes. Excess PEG and - 56 - vIruses were removed by the aforementioned wash step involving repeated sedimentation, suction and resuspension. After washed three times, 50 f.ll of PCM were added into microwells and transient gene expressions were analyzed in 48 hours. To facilitate the high-throughput analysis of expressed fluorescent proteins, the fluorescence intensities in microwells were measured by a SAFlRE plate reader (Tecan, Research Triangle Park, NC) equipped with the XFLUOR software. 4.2.4. Western blotting Protoplasts were harvested and centrifuged at 100g for 2 minutes. The pellet was resuspended in extraction buffer and mixed with 4X SDS-PAGE loading buffer. Proteins were denatured at 100°C for 5 minutes. After separation by 12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE), the proteins to be probed were electro-transferred to Immobilon-P membrane (Millipore). To detect GFP expression, the samples were then probed with the purified anti-GFP rabbit IgG at 1:2000 dilution in 2% bovine serum albumin (BSA) dissolved in TBST. After three 5-minute washes in TBST, the membrane was incubated in a secondary antibody solution containing 1:2000 alkaline phosphatase conjugated goat-anti-rabbit IgG for 45 minutes. Antibody bound green fluorescent proteins were visualized using NBT and BCIP substrates for the detection of alkaline phosphatase activity. For the protein analysis in plant tissues, 100 mg of infected leaves were frozen in liquid nitrogen and ground in 300f.l1 of protein extraction buffer consisting of 50 mM sodium phosphate buffer (pH 7.0), 5 mM p-mercaptoethanol, 10 mM EDTA and 0.1 % Triton X-IOO. The crude extract was centrifuged and the supernatant was mixed with 4X loadingbuffer for SDS-PAGE. - 57 - 4.3. Results and discussion 4.3.1. Adapt protoplast transfection in microtiter plates PEG-mediated protoplast transfection was carried out in microtiter plates in a format termed "micro-transfection" to improve the throughput of transient expression in a parallel platform. The procedure of micro-transfection is summarized in Figure 42. Plant protoplasts and recombinant TMV vectors harboring genes of interest were incubated in each microwell and exposed to high concentration of PEG for a brief period. After a simple wash step utilizing repeated sedimentation and resuspension to remove viruses and excess PEG, protoplasts were cultured in protoplast culture medium (PCM) and analyzed for transient expressions 48 hours post inoculation. The advantages of this system are as follows. First, because micro-transfection was carried out in individual microwells, a much smaller amount of viral vectors and chemicals was consumed. Therefore the costs, labors and the detrimental effects of transgenic materials to the environment were reduced. Second, it is a simple, efficient and effective way to carry out gene delivery and analyze their functions. Transient expression of GFP was detectable in 48 hours with efficiency up to 33%. Third, the system was amenable to high throughput operations since robotic systems are routinely used for processing microtiter plates. The conventional protocol of PEGmediated transfection is typically conducted in centrifuge tubes and requires repeated centrifugation to remove excess PEG and viral vector. In the micro-transfection protocol presented here, the modified washing step replaces centrifugation of protoplasts with simple dilution/gravitational sedimentation in microwells, allowing removal of a portion of the liquid media using a handmade vacuum pipette and - 58 - resuspension in fresh washing buffer. This modification enables handling of a large amount of samples and thus improves the throughput of the transfection process. Forth, recombinant proteins expressed in microwells were sufficient to be detected by Western blotting analysis, without the need to purify and concentrate samples. Figure 4-2. Flowchart of micro-transfection BY-2 protoplasts (8ul 107/mn 2/-l1 TMV or viral RNA PEG-mediated transfection in microwells (total volume=80/-ll) 70/-l140% PEG 6000 solution Incubate for 1 minute Repeat washing for three times Sedimentation for 15 minutes Remove about 3/4 ofliquid using vacuum pipettes with adaptable height Add 50/-lllwell protoplast culture medium (PCM) Analyze transient expression in 48 hours - 59- 4.3.1.1. Micro-transfection: TTOIA bGFP Recombinant TMV vector TT01A bGFP harbors the modified GFP reporter gene from plasmid pBIN m-gfp5-ER (Hase1off et al. 1997). The wild type gfp gene was mutated to remove a cryptic intron and spliced to a C-termina1 ER-retention signal HDEL (His-Asp-G1u-Leu). The gene product (mGFP5), which can be visualized under either UV or blue light excitation, is used as a simple reporter for gene expression in transgenic plants. TT01A bGFP is a hybrid tobacco mosaic virus (TMV) and. tomato mosaic virus (ToMV) vector which carries the mgfp5 gene. In vitro viral RNA transcripts of KpnI-digested TT01A bGFP were mechanically inoculated onto the lower leaves of N benthamiana plants. One week after inoculation, recombinant virions were assembled and moved systemically in the plants, producing GFP in the upper leaves (Figure 4-3, b). In some experiments, viral RNAs were encapsidated with purified TMV coat proteins and the in-vitro assembled viral particles were found to be capable of systemic infection in N benthamiana plants. When inoculating with encapsidated RNA transcripts, less amount of RNAs was needed and a higher level of GFP was produced in the inoculated plants (Figure 4-3, c). By this means the added coat proteins protected the RNAs from degrading and improved the infectivity of viral vectors. Recombinant virions from N benthamiana leaves infected with TT01A bGFP was purified and used to transfect BY-2 protop1asts in the micro-transfection format. The transient GFP expression in protop1asts was visualized under a Nikon - 60- Diaphot-TMD inverted microscope (Tokyo, Japan) under blue light illumination 48 hours post transfection (Figure 4-4). Figure 4-3. N benthamiana plants expressing GFP in systemic leaves under UV illumination. (a) Wild type, (b) Infected with TT01A bGFP, (c) Infected with encapsidated TT01A bGFP RNA transcripts. Figure 4-4. Micro-transfection of tobacco BY-2 protoplasts with TT01A bGFP. Protoplasts in microwells were observed 2 days after transfection using an inverted microscope with blue light excitation. (a) fluorescence microscopy (40x), (b) light microscopy (40x), (c) fluorescence microscopy (lOx), (d) light microscopy (lOx). - 61 - 4.3.1.2. Optimization of assay conditions Different parameters, including PEG concentration, cell age of BY-2 culture and incubation time were examined to optimize the efficiency of micro-transfection. On average, more than 200 protoplasts were counted under fluorescent microscope; and the transfection efficiency was estimated as the percentage of the number of green fluorescent protoplasts out of the total number of viable cells. The determination of protoplast viability is described in Chapter 2. PEG is able to alter the properties of the protoplast membrane and thus facilitates the uptake of viral vectors. When 40% PEG solution was added to the mixture of protoplasts and virions, the gene delivery efficiency was twice more than the transfection using 30% PEG (Figure 4-5). Figure 4-5. FlJect of PEG concentration on Micro-transfection 25% ~ u = 20% ell 'u ISell = e ~ u ~ ..='" ( lI 15% 10% 5% 1 - - - - - - - - - -- Eo-< 0% 24% 30% PEG concentration 40% Protoplasts prepared from BY-2 cells growing in exponential phase, usually four to five day after subculture, were more metabolically active and achieved higher level of transient expression (Figure 4-6). The Transfection efficiency was based on the micro-transfection using 40% PEG and examined 48 hours post transfection. - 62- Figure 4-6. Effect of cell age on Micro-transfection 30% - , . - - - - - - - - - - - - - - - - - - - - - - - - , t' I:l 25% ~ ~ 20% -f--------- ~ § 15% - f - - - - - - - - - ;: u ~ 10% 1 - - - - - - = = = - - - - I:l f Eo< 5% 0% 3 4 5 CeU age or BY·2 culture (days) To achieve high-throughput analysis of GFP expression, the fluorescent intensity of transfected protoplasts in each microwell was measured by a plate reader using excitation and emission wavelengths of 473 and 509 nm, respectively. The background fluorescence of wild type BY-2 protoplasts was also included as a control. In each condition, fluorescence intensities in triplicate samples were averaged. Parameters including incubation time of protoplasts with PEG solution (Figure 4-7) and protoplast culture period (Figure 4-8) were optimized in the micro-transfection format. Under optimal conditions, the fluorescent intensity of transfected protoplasts was twice as the control group 48 hours post-inoculation. Figure 4-7. meet ofPFGinculJation time on Miero-transfeetion 350 300 ------j 0 control. GFP expression .i' 250 .=. 200 .5 -=.. 150 E = 100 .3 ro.. 50 o IS' 30' Incubation time (minutes) - 63 - 45' Figure 4-8. Time course of Micro-transfection 400 350 1 - - - - 1 0 control • GFP expression £ ~ 300 = ~ 250 .s... = 200 ~ Col ft 150 100 Q :s 100 ri: 50 0 Day after transfection 2 4.3.1.3. Western blot analysis of expressed proteins The expression of gene products was analyzed by Western blotting using the sample preparation method modified from Valat et a1. (2000). Instead of homogenization or sonification~ protoplasts in each microwell were simply harvested by centrifugation and then resuspended in extraction buffer containing the SDS-PAGE loading dye. The total proteins were denatured at lOOoe for 5 minutes and separated by electrophoresis. The expression of GFP in infected protoplasts was detectable by Western blot using anti-GFP rabbit IgG (Figure 4-9). This result demonstrated the potential of immunoassay as a generic assay format for analyzing the gene products from the microtiter plate-based transient expression system. As an alternative to Western blotting~ enzyme linked immunosorbent assay (ELISA) may also be employed for assaying the gene products. - 64- M s 1 2 3 35kD---: 30kD--- 15kD,--- Figure 4-9. Western blot analysis of GFP expression using micro-transfection. M-RPN 800 Rainbow Marker (10 J,11), S-Recombinant GFP standard (20 ng), I-wild type protoplasts control, 2~3 -protoplasts transfected with TrOIA bGFP#7 4.3.2. Applications 4.3.2.1. Expression of red fluorescent protein (DsRed2) Red fluorescent protein DsRed, originally isolated from reef coral Discosoma striata (Matz et al. 1999), has an excitation peak at 558 run and an emission maximum at 583 run. DsRed2 is a variant of RFP that has a faster chromophore maturation rate and improved solubility relative to the wild-type protein. It is suitable for the monitoring of gene expression in living organisms and multiple labeling of tissues. A viral vector encoding DsRed2 derived from the plasmid pGDR (Goodin et al. 2002) was constructed as another reporter gene to further examine the efficiency of micro-transfection and monitor multiple gene expression efficiency. Polymerase chain reactions (peR) were used to introduce SphI and AvrIl sites (underlined) into - 65- the DsRed2 cDNA by oligonucleotide primers: DsRed2MlS (5' GCA GCA TGC CCT CCT CCG AGA ACG TCA TC 3', upstream) and DsRedL225A (5' CGT CCT AGG CTA CAG GAA CAG GTG GTG GCG GCC CTC 3', downstream). PCR was performed in a 100 III reaction mixture containing 0.1-0.2 Ilg of DNA, 0.2 mM of dNTP, 0.5 IlM of each primer, 2 units of Vent DNA polymerase and IX Vent buffer in an Eppendorf Mastercycler using tube control mode: 5 cycles at 97°C, 1 min; 55°C, 1 min; 72°C, 1 min; 20 cycles at 94°C, 1 min; 55°C, 1 min; 72°C, 1 min. The PCR product was purified by phenol/chloroform extraction and precipitated with 1/3 volume of 10M ammonium acetate and 2 volumes of 100% ethanol. For double digestion, The PCR fragment was completely digested with the SphI, which was then heat deactivated at 65°C for 20 minutes. The SphI digested DNA was precipitated with 1/3 volume of 10M ammonium acetate and 2 volumes of 100% ethanol before incubating with AvrIl. The 0.7kb SphI/AvrIl fragment of the PCR product was ligated into SphI and AvrIl digested TMV vector TTOSAI APE pBAD to create plasmid TTOSAI DsRed2 (Figure 4-10). The recombinant DNA was transformed into E. coli C600 at 42°C for 90 seconds and the precision of vector construction was confirmed by HindIlI digestion. Plasmid DNA was purified using Midiprep kit from Qiagen and used as the template for in vitro transcript reaction. - 66- pGDR peR CD RED2MlS D REDL225A) DsRed2 PCR TTOSAI APE pBAD #5 1 SphI AvrIl Phenol extraction SphI AvrIl Vector and insert purification (low melt) Ligation (subclone) Heat shock transformation C600 ITOSA1 DsRed2 Miniscreen plasmid isolation Check digestion map (HindIII) 1 1 Selected C600 colony Glycerol stock Midiprep plasmid isolation TTOSA1 DsRed2 #5 Kpnl phi Figure 4-10. Viral vector encoding red fluorescent protein: ITOSAI DsRed2. Flow chart for vector construction (top). Map ofTIOSAl DsRed2 (bottom). One week after inoculation with infectious RNA transcript of ITOSAI DsRed2~ N benthamiana plants showed symptoms such as plant stunting with mild - 67- chlorosis and distortion ofsystemic leaves (Figure 4-11, b). In Figure 4-11 (d), RFP was visualized in leaf discs of the infected plants using an Olympus BX60 fluorescent microscope with red-shifted TRITC fiher set 41002c HQ:R (HQ 545/30x, Q570LP, HQ 620/60m, Chroma Technology Corp.). Figure 4-11. N. benthamiana plants expressing RFP in systemic leaves 7 days after inoculation. (a) Wild type plant, (b) Plant infected with TTOSAI DsRed2, (c) wild type leave, RFP fiker (d) infected leave, RFP fiker. Tobacco BY-2 protoplasts were transfected by recombinant virions purified from the Nicotiana benthamiana plants infected with TTOSAI DsRed2, using the micro-transfection system developed and optimized before. Transient RFP activities were detected using a Nikon inverted microscope under green light illumination 48 hours post inoculation with efficiency of5% (Figure 4-12). - 68- Figure 4-12. Micro-transfection of tobacco BY-2 protoplasts with TrOSAI DsRed2. Protoplasts in microwells were observed 2 days after transfection using an inverted fluorescence microscope with green light excitation. (a) BY-2 protoplasts, (b) BY-2 m-gip5-ER protoplasts. 4.3.2.2. Expression of rice a-amylase in micro-transfection system A hybrid tobamoviral vector TrOIA 103L (Kumagai et aI. 2000) encoding rice a-amylase was used as a model to express secreted recombinant protein by micro-transfection. The cDNA was placed under the control of the ToMV subgenomic promoter in TrOIA and spliced to a rice a-amylase signal peptide sequence to target the rice a-amylase for secretion. Protoplasts were transfected with 0.2 ~g of infectious RNA obtained by in vitro transcription of the plasmid TrOIA 103L. Two days post transfection, supernatants were collected from micro-wells and mixed with the amylase reagent (Sigma kit #568-20). The rice a-amylase activity was calculated as the difference in OD40S measured by a plate reader, which was used to improve the throughput of analysis. High level of amylase activity was expressed in transfected protoplasts after 18-hour incubation (Figure 4-13). - 69- Figure 4-13. a-amylase activity in transfected protoplasts 2.5 o control 0 2 amylase activity I - ~ 1.5 ~ o - '" 0.5 o i--- - ---I - - - I 2 Bours after assay 18 4.3.2.3. Expression of multiple proteins Two strategies were explored for transient expression of multiple proteins in plants or plant protoplasts. In one strategy, the hosts are inoculated with a mixture of recombinant viruses harboring different genes. The other strategy is to clone cDNAs carrying various gene cassettes in one vector and insert internal ribosome entry site (IRES) or linker sequences, such as Kex2 or Tobacco Etch Virus (TEV) protease recognition sites, among cDNAs. After the translation, proteins will be expressed separately. The transient expression of multiple proteins in a host by single or multiple viral vectors is a useful tool for protein interaction studies. 4.3.2.3.1. Dual inoculation To explore the possibility of transfection with more than one construct, both viral vectors TTOIA bGFP and TTOSAI DsRed2 were transferred into BY-2 protoplasts via micro-transfection (co-transfection) or onto the lower leaves of N benthamiana by mechanical inoculation (dual inoculation). From Figure 4-14 and 4-15, both fluorescent proteins were visible in infected -70 - protoplasts and systemic leaves of plants. The expression levels of these proteins were varied depending on the infectivity of each viral vector. For protein interaction studies that require equivalent amounts of expressed proteins, the strategy of multiple gene expression by a single vector is more suitable. Figure 4-14. Co-transfection ofBY-2 protoplasts with (a) TTOlA bGFP and (b) TIOSAI DsRed2. 2 days after micro-transfection, same protoplast was viewed under a fluorescence microscope using filters for GFP and RFP, respectively. Figure 4-15. Dual inoculation of N. benthamiana with TIOlA bGFP and TTOSAI DsRed2. Six days post inoculaiton, systemic leaves were visualized under a fluorescence microscope using filters for GFP and RFP, respectively. 4.3.2.3.2. Multiple gene expression by a single vector To demonstrate the second strategy of multiple gene expression, a hybrid TMV vector encoding two fluorescent proteins connected by Kex2p linker - 71 - sequence (TTOSAI RFPKexGFP) was constructed. Kex2p is the prototype of a Golgi-resident protease responsible for the processing of prohormones in yeast and mammalian cells. Kex2p-like protease activity was found in the trans-Golgi network (TGN) of plant cells (Jiang and Rogers, 1999). Brefeldin A (BFA) which blocks the trafficking of proteins beyond the cis-Golgi (Klausner et aI., 1992) prevents Kex2 cleavage in the reporter. Once the recombinant virus replicated in infected plants, the fusion protein containing GFP and DsRed2 was synthesized in ER and transported to the Golgi apparatus. Cleavage of the fusion protein in Golgi by Kex2 protease leads to the expression of separate proteins. This strategy is feasible to express multiple gene products, monitor expression level of interested genes using accompanying reporter genes and study protein interactions. 4.3.2.3.2.1. Construction of TTOSAI RFPKexGFP A two-step cloning strategy was used to create a fusion protein of DsRed2 and GFP linked by the Kex2 sequence (Figure 4-16). Methods for plasmid construction and PCR conditions were described previously. The Kex2 coding sequences (GGIGKRGKIGKRGKIGKRGKEF) containing three tandemly repeated Kex2 cleavage sites were derived from the construct pLJ607. GG residues are spacers to permit flexibility, and EF is the introduced EeoR! site. Two oligonucleotide primers, mGFPS2E (5' GCC GAA TTC AGT AAA GGA GAA GAA CTT TTC 3', upstream) and mGFPL242AE (5' GCG GAA TTC CCT AGG TTA AAG CTC ATC ATG TTT GTA TAG 3', downstream) were synthesized and used for PCR - 72- amplification of pBIN m-gfp5-ER. The 750bp EcoRI digested fragment was cloned into pLJ607 to generate an intermediate vector pLJ607 mGFP5. The DNA fragment encoding Kex2 cleavage sites and GFP was amplified from this vector by PCR using the following primers: LJ607R4X (5' GCA CTC GAG GCA GGA GGA ATA GGC AAA CGG 3', upstream) and mGFPL242AE (5' GCG GAA TTC CCT AGG TTA AAG CTC ATC ATG TTT GTA TAG 3', downstream). The incorporatedXhoI and AvrIl sites were underlined, while the C-terminal ER-retention signal HDEL (His-AspGlu-Leu) was in bold characters. For double digestion, DNA was completely digested with the first enzyme, which was then heat deactivated. The digested DNA was precipitated with ethanol before incubating with the second enzyme. Another PCR was carried out to introduce SphI and XhoI sites (underlined) into the DsRed2 cDNA by oligonucleotides: DsRed2M1S (5' GCA GCA TGC CCT CCT CCG AGA ACG TCA TC 3', upstream) and DsRed2HDELX (5' GCT CTC GAG CTC ATC ATG CAG GAA CAG GTG GTG GCG 3', downstream). A three-piece ligation was performed to clone these two PCR fragments into viral vector TTOSAI I03SP EK CD43, that had been digested with SphI and AvrIl, to yield the plasmid TTOSAI RFPKexGFP (Figure 4-17). All these resulted in the following chimeric fusion protein: rice a-amylase signal peptide-SphI-DsRed2-HDEL-XhoIKex2 recognition site-EcoRI-mGFP5-HDEL-AvrIl. The recombinant DNA was transformed into E. coli C600 using heat shock method and the precision of the cloning was confirmed by restriction mapping and DNA sequencing. - 73 - pLJ607 pBIN ml-7i~::s2E' mGFPL242AE) mGFP5-HDEL PCR EcoRI L..-----r-----------l Phenol extraction EcoRI Insert purification (gel extraction) Ligation (subc1one) Transformation C600 pLJ607 mGFP5 Miniscreen plasmid isolation Check digestion map (Sad, EcoRI) PCR analysis 1 Selected C600 colony ! Glycerol stock (log #250) Miniprep plasmid isolation TTOSAI l03SP EK CD43PU607 mlG~;~R4X' mGFPL242AE) Kex2-mGFP-HDEL PCR SphI AvrIl PGID~:RED2M1S' DSRED2HDELX) DsRed2-HDRL peR Phenol extraction XhoI AvrIl Phenol extraction SphI XhoI Vector and insert purification Ligation (subc1one) Transformation C600 TTOSAI RFPKexGFP ! ! Miniscreen plasmid isolation Check digestion map (XhoI) Selected C600 colony Glycerol stock (log #256) Midiprep plasmid isolation TTOSAI RFPKexGFP #4 Figure 4-16. Flowchart for vector construction: TTOSAI RFPKexGFP. - 74- Figure 4-17. Plasmid map of ITOSA1 RFPKexGFP. 4.3.2.3.2.2. Expression of fusion fluorescent protein In vitro RNA transcripts from viral cDNA clones were mechanically inoculated onto N benthamiana plants, causing a systemic infection in one or two weeks. Both GFP and DsRed2 were visualized in the leave discs of the infected plant under a fluorescence microscope with corresponding filter sets (Figure 4-18). Western blot analysis was performed to determine the size of expressed fluorescent proteins using primary antibodies specific for DsRed and GFP, respectively. By comparing with the molecular weight marker and standard individual fluorescent proteins, the majority of transient gene products were found to have a molecular weight of about 60 kDa, indicating that fusion proteins ofDsRed2 and GFP were expressed (Figure 4-19). - 75- Figure 4-18. Infection of TTOSAI RFPKexGFP #4 in systemic leaves of N. benthamiana. Ten days post inoculation, a portion of leave discs was observed under an Olympus BX60 microscope. (a) Light microscopy, (b) Fluorescence, SWB filter, (c) Fluorescence, RFP filter, (d) Fluorescence, GFP filter. G 75kD- s 75kD .. .. - 50kD- R s 50kDkD- 30kD- 35kD30kD- Figure 4-19. Western blot analysis of extracts infected by TTOSAI RFPKexGFP #4. Proteins were probed with anti-GFP rabbit IgG (left) and living color DsRed monoclonal antibody (right), respectively. M-Rainbow molecular weight marker, G-Recombinant GFP standard, R-DsRed2 standard, S-sample extracts. -76 - This result was not what we expected because few separate RFPs or GFPs were expressed. We went over all the details of the protocols and drew a conclusion that it was because of an inappropriate design of the vector. When the viral vector was constructed, a C-terminal ER-retention signal HDEL (His-Asp-Glu-Leu) was incorporated into the oligonucleotides for the PCR of both DsRed2 and GFP fragment. Thus during the infection of the recombinant virus, the fusion proteins synthesized in ER were probably retrieved to ER by the HDEL receptors in cis-Golgi reticula and could not reach the plant Kex2p sites in trans-Golgi network. Thus another construct without HDEL signal was proposed to solve this problem but was not included in this thesis. 4.4. Concluding remarks Viral transfection of plant protoplasts has been used for transient expression studies for a long period oftime. However, this method has not been reported to be carried out in a small volume and, therefore, potential for the high-throughput expression and analysis of a large amount of genes simultaneously. We modified the protocol and conditions of regular transfection method and developed the micro-transfection system in this chapter. In micro-transfection, protoplasts were transfected by viral vectors in 96-well plates. Several modifications were adapted to improve the throughput of transient expression, such as modified washing step instead of centrifugation, using a plate reader to measure fluorescence or absorbance in samples, using encapsidated RNA transcripts instead of purified virions. Assay conditions were optimized based on the expression efficiency using GFP reporter gene. The transfection efficiency reached as high as 33% in 48 hours. - 77- A variety of viral vectors were constructed and used as models to demonstrate the transient expression of single or multiple gene products, secreted recombinant proteins and fusion proteins. Two strategies were explored to express multiple gene products by single or multiple viral vectors and facilitate protein interaction studies. To demonstrate the first strategy, TMV vector encoding green and red fluorescent proteins were mixed and transferred into the hosts. The other strategy was to express multiple proteins by one vector. A viral vector encoding two fluorescent proteins connected by the Kex2 recognition sequences was constructed. However, the presence of the C-terminal ERretention signal HDEL in the viral vector prevented the gene products from cleavage by Kex2 protease. Thus the removal of HDEL is suggested for the characterization of Kex2p activities in plant cells and the separation of multiple proteins. 4.5. References Bailey S.N., Wu R.Z., Sabatini D.M., 2002. Application oftransfected cell microarrays in high-throughput drug discovery. Drug Discovery Today 7 (18): 1-6. Goodin M.M., Dietzgen R.G., Schichnes D., Ruzin S., Jackson A.G., 2002. pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. The Plant Journal 31 (3): 375-383. Gooding G.V., Herbert T.T., 1967. A simple technique for purification oftobacco mosaic virus in large quantities. Phytopathology 57: 1285. Haseloff J., Siemering, K.R., Prasher D.C., Hodge S., 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the National Academy of Sciences of USA 94: 2122-2127. Jiang L.W., Rogers J.C., 1999. Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells. The Plant Jouma118 (1): 23-32. - 78- Klausner R.D., Donaldson J.G., Lippincott-Schwartz J., 1992. Brefeldin A: insights into the control of membrane traffic and organelle structure. Journal of Cell Biology 116: 1071-1080. Koop H.D., Steinmiiller K., Wagner H., RoBler C., Eibl C., Sacher L., 1996. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Planta 199: 193-201. Kumagai M.H., Donson J., Della-Cioppa G., Grill L.K., 2000. Rapid, high-level expression of glycosylated rice a-amylase in transfected plants by an RNA viral vector. Gene 245: 169-174. Matz M.V., Fradkov A.F., Labas Y.A., Savitsky A.P., Zaraisky A.G., Markelov M.L., Lukyanov S.A., 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnology 17: 969-973. Valat L., Toutain S., Courtois N., Gaire G., Decout E., Pinck L., Mauro M.C., Burrus M., 2000. GFLV replication in electroporated grapevine protoplasts. Plant Science 155: 203-212. - 79- CHAPTER 5. MICRO-TRANSFORMATION 5.1. Introduction In addition to the aforementioned transient expression systems based on protoplast transfection by viral vector, we adapted another gene transfer method, Agrobacteriummediated transformation of plant cells, into microtiter plates in this chapter. This system was termed micro-transformation and was amenable for high-throughput transient gene expression in plant cells even though no report has been made previously. 5.1.1. Agrobacterium-mediated transformation Agrobacterium-mediated transformation is widely used to create transgenic plants in modem plant biology and agricultural biotechnology (Gelvin, 2003; Li et aI., 2000). Agrobacterium tumefaciens is a soil bacterium which can cause the formation of crown galls or tumors in many plant species. The ability of Agrobacterium to transform plants comes from virulence genes found on the vir region of the tumorinducing (Ti) plasmid. The expression of virulence gene is induced by the presence of acetosyringone, a phenolic compound usually released from wounded plant cells. During invasion a portion ofTi plasmid called transferred DNA (T-DNA) is delivered and stably integrated into the plant genome. T-DNA is bound by two 25bp imperfect repeat sequences called right and left border and encodes genes for the synthesis of metabolites during tumor induction such as opines, auxin and cytokinin. Removal of these genes leads to a disarmed Agrobacterium strain that is no longer oncogenic. Agrobacterium binary vectors are used to introduce foreign genes into plant cells (An, 1985; Rempel and Nelson, 1995) or protoplasts (Marton, 1984; Depicker et aI., - 80- 1985) by molecular biologists. In a binary vector, the two main components for essential tumor inducing, T-DNA and the vir region, are located on separate plasmids (Hoekema et al. 1983, Hellens et al. 2000). The vir gene resides on the helper Ti plasmid of the disarmed Agrobacterium strain; while the T-DNA on the binary Ti plasmid is genetically manipulated to harbor foreign genes to be transferred into the hosts. Dominant selectable marker genes are also introduced in the recombinant vector to select and maintain the binary vectors. Several transformation approaches include coculture of plant cells or protoplasts, leaf discs, seeds or other tissues with the bacteria, vacuum infiltration and biolistic methods. 5.1.2. Transient expression system based on tobacco cells A transient expression system termed micro-transformation was derived from the transformation of tobacco cells using Agrobacterium binary vector in 24-well culture plates. In the model system, tobacco BY-2 cells were co-cultivated with Agrobacterium tumefaciens binary vector harbored with the GFP expression cassette. After three or four days, transient GFP expression was analyzed using fluorescence microscopy, fluorescent plate reader and Western blot. Different parameters including Agrobacterium concentration, acetosyringone, light and temperature were optimized based on the transformation efficiency. The precision and consistency of this system were estimated by intra-assay and inter-assay coefficient of variations (CV). 5.2. Materials and Methods 5.2.1. Chemicals Murashige and Skoog (MS) basal medium salt mixtures were purchased from Phyto Technology Laboratories (Shawnee Mission, KS). Kanamycin and carbenicillin - 81 - were purchased from Fisher Scientific Co. (Houston, TX). Geneticin G 418 was obtained from Agri-Bio (North Miami, FL). 5.2.2. Agrobacterium binary vector Plasmid pBIN m-gfp5-ER (Haseloff et al. 1997) encoding modified GFP was electroporated into Agrobacterium tumefaciens strain C58Cl in a 0.2 cm electroporation cuvette using Bio-Rad Gene Pulser II apparatus set at 2.2 kV, 4000 and 25 JlF. Transformed binary vectors were grown on selective YM solid media containing 0.04% yeast extract, 0.1% mannitol, 1.7 mM NaCl, 0.8 mM MgS0 4·7HzO and 2.2 mM KzHPOdHzO (pH 7.0), 1% agar, 100 Jlg/ml of kanamycin and 5 Jlg/ml of tetracycline. 5.2.3. Agrobacterium-mediated transformation 5.2.3.1. Transformation of BY-2 cells Tobacco BY-2 cells were cultured in MSD medium containing 43 gil MS salt, 0.2 gil KHZP04, 1 mg/l thiamine-HCI, 0.2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 gil myo-inositol and 3% sucrose, pH5.8. Three days after subculture, 2 ml of cells were transferred to a 35 mm Petri dish or 6-well culture plate and cocultured with 50 JlI of overnight culture of Agrobacterium tumefaciens C58Cl harboring pBIN m-gfp5-ER in dark at room temperature for 72 hours. 10 JlI of acetosyringone stock solution (20mM in ethanol) were added per ml of cells. Transient GFP expression was monitored using an Olympus BX60 fluorescent microscope with U-M41017 endow GFP filter (HQ470/40, Q4951p, HQ525150, Chroma Technology Corp.). The transformation efficiency was estimated as the - 82- ratio of the number of green fluorescent cells to the total number of viable cells. Cells were then washed with fresh medium for three times and plated on solid medium containing 10 /-lg/ml of G418 and 500 /-lg/ml of carbenicillin for selection. For micro-transformation, 400 /-ll of BY-2 cells were mixed with 10 /-ll of Agrobacterium solution in a Falcon 24-well plate. Acetosyringone was added to a final concentration of 200 /-lM. The plate was wrapped with aluminum foil and incubated at room temperature for three to four days. The fluorescence intensity in each well was analyzed by a SAFIRE plate reader (Tecan, Research Triangle Park, NC) using excitation and emission wavelengths of 473 and 509 nm, respectively. To reduce standard errors, duplicate samples were measured using the multiple reading function (4x4 circle) provided by the XFLUOR software. 5.2.3.2. Transformation of BY-2 protoplasts The isolation and culture of BY-2 protoplasts were described in chapter 2. After isolation, protoplasts were allowed to regenerate their cell walls for three days. 2 ml of protoplast solution containing 200 /-lM of acetosyringone were mixed with 50 /-ll of overnight culture of Agrobacterium tumefaciens e58Cl harboring GFP or GUS reporter gene in a 35-mm Petri dish. The co-cultivation was carried out in dark at room temperature for 72 hours. Transient expression of GFP and GUS was analyzed by fluorescent microscopy and GUS assay, respectively. 5.2.4. Western blotting Three to four days after co-cultivation, 100 /-ll of transformed BY-2 cells were harvested by centrifuging at 1000 rpm for one minute. The cell pellets were - 83 - resuspended in GFP extraction buffer and mixed with 4X SDS-PAGE loading buffer. Proteins were denatured at 100°C for 5 minutes. After separation by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), the proteins to be probed were electro-transferred to Immobilon-P membrane (Millipore). The expression of GFP in transformed cells was detected by Western blot using anti-GFP rabbit IgG and alkaline phosphatase conjugated goat-anti-rabbit IgG. 5.2.5. GUS activity assay The j3-glucuronidase (GUS) activities in transformed protoplasts were analyzed using the histochemical method developed by Jefferson (1987). 500 J..lI of protoplast solution was washed and mixed with 300 J..lI of staining solution containing 0.5 mg/ml of 5-bromo-4-chloro-3-indolyl gulcuronide (X-Glue), 10% dimethylformamide (DMF) in 50 roM sodium phosphate buffer (pH 7.0). After an overnight incubation at 37°C, transformed protoplasts were stained blue and visualized under light microscope. 5.3. Results and discussion 5.3.1. Micro-transformation A transient expression system termed micro-transformation was developed based on the Agrobacterium-mediated transformation of tobacco BY-2 cells in 24-well plates. A. tumefaciens LBA4404 or C58Cl harbored with binary vectors carrying the GFP or GUS reporter gene was used to demonstrate the advantage of this system. First, although BY-2 protoplasts were able to be transformed by Agrobacterium and expressed transient GFP or GUS, the transformation using BY-2 cells was more efficient and simpler (Figure 5-1, 5-2). The use of cells instead of protoplasts eliminates the efforts to isolate protoplasts and maintain a good quality of the - 84- protoplast culture. Second, higher transfurmation efficiency was achieved with BY-2 cells (up to lOOI'o) than protoplasts. This was probably because the cell culture was more active metabolizing, which fiwilitated the uptake and integration of the T-DNA carrying fureign genes. Third, transfurmation in the 24-well furmat is amenable fur high-throughput analysis of gene expression. Parallel comparison of different conditions or vectors is feasible. Three or fuur days after micro-transfurmation, transient GFP expression was detectable by a fluorescence plate reader with acceptable variation. Last, the incorporation of anttoiotic resistance markers allowed the future selection of transfurmed cells. After screening fur transient expression, transfunnants with better strait can be directly selected to generate a stable cell line. Figure 5-1. Agrobacterium-mediated transfunnation. (a) BY-2 protoplast expressing GFP, (b) GUS activity in transfurmed protoplast, (c) BY-2 cells expressing GFP. Figure 5-2. Micro-transfurmation ofBY-2 cells with Agrobacterium C58Cl m-gip5ER. Cells were visualized three days after co-cultivation under blue light excitation using a Nikon inverted microscope. - 85- 5.3.2. Optimization of assay conditions Different parameters including cell density, Agrobacterium concentration, acetosyringone, light and temperature were optimized based on the transformation efficiency using Agrobacterium binary vector encoding modified GFP. Tobacco BY2 cell suspension is a fast growing and homogeneous culture. Three days after subculture, the culture was in exponential phase when most single cell clumps contain more than 16 cells. The rapid cell growth rate of the culture caused high transformation frequency and high level of transient expression. 400 III of the threeday-old BY-2 cells at a density of 1 to 2x105 cells/ml were used as the host for individual micro-transformation in 24-well plate. Agrobacterium tume/aciem strain C58C 1 containing binary plasmids was grown in YM media with appropriate antibiotics. After incubated on rotary shaker at 28°C for overnight, the bacterial culture reached an absorbance of 0.3 to 0.5 at 600 nm. From figure 5-3, at least 10 III of the Agrobacterium solution was used for transformation. 200 IlM of acetosyringone was added to the media for co-cultivation to induce the transcription of vir gene and facilitate the transformation (Godwin et al. 1991). Figure 5-3. Micro-transformation: Agro. concentration 200 . -Il""""""""""""""""""=-O.------------....------, ~ 180 11:1 ~ .51 160 e 140 e !c 120 11:1 ::I ilO: 100 80 .L.------..,.......--.. . . .- - - r - - - - - - - - - - - - - \ 2.5 3 3.5 4 .5 Day after tran formAtion - 86- s S.S 6 During the co-cultivation, the plates were wrapped with aluminum foil although light seemed to have no significant effects on transient expression of GFP. A larger percentage of green fluorescent cells were observed when the transformation was taken place at room temperature instead of 26°C. The optimal transient GFP activities were detected after 3 or 4 days of co-cultivation with efficiency up to 10%. To facilitate the high-throughput analysis of transient GFP expression, the fluorescent intensity of transformed cells in individual microwell was measured by a multiple plate reader using excitation and emission wavelengths of 473 and 509 nm, respectively. The background fluorescence of wild type BY-2 cells was included as a control. In each well, the fluprescence intensity was averaged from readings of 12 spots (4x4 circle function provided by the XFLUOR software). For each condition, at least two samples were analyzed to minimize the standard errors. By this way, the GFP activities in transformed cells were monitored every day (Figure 5-4). Four days after transformation, the green fluorescence increased to a level that could be distinguished from the control. In five days the GFP expression increased to twice as high as the auto-fluorescence. Figure 5-4. Time course of micro-transformation 3 5 0 . , . . . - - - - - - - - - - - - - -_ _,...,.. C 300 ~ Control - -, GFp expression .; 250 .fl .51 200 .... ~ 150 u E = 100 ;= 50 2 3 4 S Day after lnoculation - 87- 6 7 8 The micro-transformation ofGFP was confirmed by Western Blotting (Figure 55). The total proteins extracted from 30 J,11 of BY-2 cells were denatured and separated by SDS-PAGE, followed by electro-transferred to a polyvinylidene fluoride (PVDF) microporous membrane. GFP which has a molecular weight of about 29 kD was probed with anti-GFP rabbit IgG and detected by chemi-Iuminescent reagents. M 1 2 3 Figure 5-5. Western blot analysis of micro-transformation. M-Rainbow Molecular Weight 35kD- Marker, 30kD- I-Wild type BY-2 cells, 2-3-BY-2 cells transformed with Agrobacterium C58Cl mgfp5-ER. 5.3.3. Assay quality control The precision and consistency of this system were estimated by intra-assay and inter-assay coefficient of variations (CV) (Athar et aI., 2004; Murray et aI., 1993). Intra-assay CV is the ability of the assay to consistently reproduce a result when samples are taken from the same treatment. The fluorescent intensities of 11 duplicate micro-transformation smnples carried out on the same plate were analyzed and calculated as follows. A figure of 10% or less is considered satisfactory. Mean of the Standard Deviations of the Duplicates Intra-assay CV x 100% Grand mean of the duplicates - 88- To ensure the results obtained from different time and batches are consistent and comparable, inter-assay CV were estimated from 9 individual determinations as follows. A reasonable target for %CV in routine testing is 10-15%. Standard Deviation of the means of the duplicates Inter-assay CV x 100% Grand mean of the duplicates Based on the above formulas, the Intra-assay and Inter-assay CV for microtransformation are 3.74% and 14.08%, respectively. Each BY-2 sample was assayed after four days of co-cultivation with A. tumefaciens C58C1 pBIN m-gfp5-ER in dark. 5.4. Concluding remarks Agrobacterium-mediated transformations of BY-2 cells were carried out in 24-well plates with reduced amount of hosts and inoculums. To demonstrate the application of this so-called micro-transformation system, GFP reporter genes were expressed in BY-2 cells with efficiency up to 10% in four days. The GFP expression level was determined using a fluorescence plate reader to facilitate the analysis. The readings showed reasonable consistence and reproducibility when negative controls were included. Because of its efficiency and effectivity, micro-transformation is recommended for highthroughput transient analysis of gene products and parallel comparison or screening of different vectors or conditions in future experiments. - 89- 5.5. References An G., 1985. High efficiency transformation of cultured tobacco cells. Plant Physiology 79: 568-570. Athar H., Iqbal J., Jiang X.C., Hussain M.M., 2004. A simple, rapid and sensitive fluorescence assay for microsomal triglyceride transfer protein. Journal of Lipid Research 45: 764-772. Depicker A., Herman L., Jacobs A., Schell J., Van Montagu M., 1985. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Molecular and General Genetics 201: 477484. Gelvin S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiology and Molecular Biology Reviews 67 (1): 1637. Godwin I., Todd G., Ford-Lloyd B., Newbury H.J., 1991. The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species. Plant Cell Reports 9: 671-675. Haseloff J., Siemering, K.R, Prasher D.C., Hodge S., 1997. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the National Academy of Sciences of USA 94: 2122-2127. Hellens R, Mullineaux P., Klee, Harry, 2000. A guide to Agrobacterium binary Ti vectors. Trends in Plant Sciences 5: 446-451. Hoekema A., Hirsch P.R., Hooykaas P.J.J., Schilperoort RA., 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179-180. Jefferson RA., 1987. Assaying chimeric genes in Plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5: 387-405. Li W., Guo G., Zheng G., 2000. Agrobacterium-mediated transformation: state of the art and future prospect. Chinese Science Bulletin 45 (17): 1537-1546. - 90- Marton L., 1984. Transformation oftobacco cells by coculture with Agrobacterium tumefaciens. Cell Culture and Somatic Cell Genetics of Plants 1: 514-521. Murray W., Peter A.T., TeclawR.F., 1993. The clinical relevance of assay validation. Compendium on Continuing Education for the Practicing Veterinarian 15 (12): 1665-1675. Rempel H.C., Nelson L.M., 1995. Analysis of conditions for Agrobacterium-mediated transformation oftobacco cells in suspension. Transgenic Research 4: 199-207. - 91 - CHAPTER 6. CONCLUSION AND RECOMMENDATION Based on the studies using different transient expression formats to improve the throughput of gene transfer, several conclusive remarks were listed as follows. 1. Isolation and purification of tobacco protoplasts, molecular cloning of recombinant TMV vectors, viral protoplast transfection, Agrobacterium-mediated transformation oftobacco cells and protein assays were the basic techniques involved in this thesis. 2. Four transient expression formats were developed and evaluated: well-less cell array system including single and double layer hydrogels, micro-transfection and microtransformation. 3. The cell immobilization or entrapment technique using polycation-coated alginate gels was established. Although the well-less cell array system was novel, simple and amenable for high-throughput transient expression, the efficiency of transfection was not sufficient for detection of expressed genes because of some technical obstacles. 4. Two microtiter plate-based transient gene expression systems based on different hosts and gene delivery approaches were developed. Assay conditions were optimized based on the transient expression of GFP as a reporter gene. The summaries of these systems were listed in the Table 6-1. These transient expression formats, which have not been reported before, are attractive for highly parallel gene expression which could accelerate studies of gene functions, protein interactions and drug screening, at reduced scales and costs. 5. As a prerequisite for studying protein interaction using viral vectors, the strategies to express multiple proteins in plants or plant protoplasts were explored. In dual - 92-