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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.
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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
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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.
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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.
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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
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- 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.
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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
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