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Molecular methods of cell culture II Expression of mammalian expression vector in cell culture -Galactosidase Reporter system Hydrolaze enzyme catalyze hydrolysis of -galactoside to monosaccharide Contain 1023 a.a. Mammalian -Galactosidase Reporter system Expression of -Galactosidase in WI-380 cells Tet on Inducible system Induction by Doxicyclin TRE: Tetracyclin( Doxicyclin) response element rtTA : regulatory protein ubiquitous promoter (PU) or tissue- or cell type-specific promoter (PTS) Gene of interest is under the regulation of the rtTA-dependent promoter Acta Biochimica et Biophysica Sinica 2007, 39(4): 235–246 Expression of Fluorescent protein in cell culture Green fluorescent protein ( GFP) 238 a.a or Red Fluorescent protein Cloned from jelly fish( Aequorea victoria) Red Fluorescent protein fusion protein to the multiple cloning site GFP DAPI HEL: Human Embryonic Lung RFP DAPI HEL: Human Embryonic Lung Promoter reporter system Luciferase assay MCS SV40 Luciferase Multiple cloning site Luciferase gene NON-VIRAL GENE DELIVERY 1. Synthetic Polymers To generate cationic polymers to interact electrostatically with and neutralize negatively charged DNA Poly L-lysine (PLL) high cytotoxicity aggregate and precipitate polyethylene glycol (PEG) flexible, water-soluble ----Polymer covalent coupling of PEG( PEGylation) of a target molecule i.e. PLL : cytotoxicity and non-specific protein adsorption i.e. polyethyleneimine (PEI), a cationic gene carrier with superior transfection efficiency and unique buffering properties 2. Natural Polymers cyclodextrin, chitosan, collagen, gelatin, and alginate Advantage: Innate environmental responsiveness Ability to be degraded Remodeled by cell-secreted enzymes. Non-toxic at both low and high concentrations, are Readily incorporated into oral or bolus matrix delivery systems, Serve as tissue engineering scaffolds Entry of DNA-Polymer Complex DNA :polymer complex DNA :polymer complex DNA :polymer complex endosome Episomal or integration with selection Transcription Virus vector Gene delivery in cell culture system Deliver vector for gene therapy Recombinant protein production in industry Clinical application of viral vector Adenovirus( Tumors, Haemopoietic cells AAV( liver, muscle, retinal Polymer coated adenovirus ( tumors) Retrovirus( t umor,stem,h aemopoitic cells Herpes simplex virus(CNS, PNS,muscle, Haemopoiet ic, stem cells Alpha virus ( tumors) Lentivirus( CNS, liver,muscl e Liposome encapsula te alpha virus TRENDS in Biotechnology Vol.21 No.3: 119 2003 Requirements for viral gene therapy vectors 1. Delivery system must be safe and immunologically inert. 2. Protect the genetic material from degradation. 3. Vector must encode an effective therapeutic gene that has sustained expression at a defined target site 4. Tissue-specific targeting 5. Site-specific chromosomal integration 6. Controlled infection of both dividing and non-dividing cells Virus Vectors retrovirus Recombinant cell lines SV-40 substitute virus gene with foreign genes ( supply virus missing gene by cotransfection with helper virus) Infect monkey cells only Carry smaller size of foreign genes SV-40 T ag integration T ag protein allows replication of plasmid Vaccinia virus Carry smaller size of foreign genes DNA recombination occurs in the cells Virus replicate within the cytoplasm of the host cells Higher level of protein expression Baculovirus Foreign gene maybe coexpressed with structural gene ( structural protein expresses when infection occurs) replacement of coat protein gene with gene of interest Baculovirus Replace coat protein gene with gene of interest Developing baculovirus-insect cell expression system for humanized recombinant glycoprotein Baculovirus-insect cell expression system 4. Retrovirus Recombinant retroviruses used in gene delivery: 1.γ-retroviruses 2. Lentiviruses 3. spumaviruses Retrovirus life cycle Retroviral vector development for increased efficiency and targeting Structure of a simple retroviral genome containing coding sequences for gag, pro, pol, and env for replication att att U3 R U5 PBS gag pro pol env 5’LTR PPT U3 R U5 3’LTR Genome 7-10kb contain gag, pro, pol, env : encode structural capsid proteins, viral protease, integrase, and viral reverse transcriptase, enveloped glycoproteins Retroviral vector development for increased efficiency and targeting att U3 att att R U5 PBS gag pro pol env PPT U3 5’LTR U3 R 5’LTR U5 PBS R U5 att 3’LTR cPPT Transgene U3 R U5 P ampr 3’LTR Replication coding sequences removed and transgene inserted. U3 component of the 5′LTR is used as a promoter to drive transgene expression P: Heterologous or tissue-specific promoter inserted to drive an ampicillin gene to facilitate ex vivo selection of transduced cells. LTRs: dual long-terminal repeats , (PBS): primer binding site , Ψ: signal, (att): attachment sites polypurine tract : (PPT). Retroviral vector development for increased efficiency and targeting Structure of an enhanced self-inactivating retroviral gene therapy vector att U3 att att R U5 PBS gag pro pol env PPT U3 5’LTR RSV R U5 PBS 5’LTR R U5 att 3’LTR cPPT P Transgene CI,CR,WPREs/MAR,ampr substitution of the 5′LTR U3 component by RSV. U3 R U5 3’LTR P: internal promoter is tissue-specific to limit transgene expression. Additional genetic elements—incorporated to enhance site specificity and integration efficiency while limiting CI : chromatin insulators , limit position effect variegation CR: chromatin structure regulators WPRE : woodchuck hepatitis virus post-transcriptional regulatory element, enhance mRNA transcript stability S/MAR : scaffold or matrix attachment regions or resistance, anchorage of chromatin with stabilization of chromosomal loops Function of 3rd generation MLV packing cell line Therapeutic gene Stable integration Advantages of retrovirus vector Stably traduce dividing cells Long term transgene expression RNA virus ( virus genome may be integrated into the host genome) Infect various kinds of mammalian cell lines Infection of mammalian cells by retrovirus does not cause host death Carry -galactosidase gene Viral gene expression is driven by stronger promoter Low immune response Disadvantage of retrovirus vector Low viral titer Only infecting dividing cells Integrative may activate or damage cellular genes Random insertion into host cell and causes oncogenic activation or tumor-suppressor gene Limited insert capacity( 8kb) Inactivation by human complement Inability to transduce nondividing cells Lentivirus human immunodeficiency virus (HIV) simian immunodeficiency( SIV) non-primate- equine infectious anaemia virus (EIAV) feline immunodeficiency virus (FIV) Recombinant lentiviral (rLV) vector systems tool to achieve high transduction efficiency for non-dividing cells e.g. central nervous system, retinal cells, pancreatic islets, progenitor and differentiated hematopoietic cells Accessory and Regulatory genes oncogenesis (tat), apoptosis (vpr), MHC downregulation, (nef ) differentiation (vpu) Biologically active and can cause detrimental effects on cells Lenti virus vectors lacking tat, vpr, nef vpu TU, transducing units; p, promoter; env, envelope; WPRE, Woodchuck hepatitis posttranscriptional regulatory element; cPPT, central polypurine tract; CMVp, cytomegalovirus promoter Lentiviral transduction of quiescent T-cells The T-cell-specific stimulation then allows reverse-transcription of the viral genome followed by nuclear import and integration of the proviral DNA J Gene Med 2004; 6: S83–S94. Retroviral Transduction of T-cell Receptors in Mouse T-cells http://www.jove.com/details.php?id=2307 Adeno virus Largest non-enveloped virus and contain linear, double-stranded Genome :36kB ITR Early E1A Late ITR Early genes : function as regulatory proteins for viral replication Late genes : encode structural proteins for new virus assembly E1A gene: a trans-acting transcriptional regulatory factor that is required for early gene activation CAR receptor pH dependent release of virus particle Adenovirus Serotype Group C: most common in nature and in adenoviral gene therapy are human serotypes 2 and 5 Genome type of Adenovirus and different types of Ad5 derived vector E1A E1B E3 MLP L1 L2 L3 L4 L5 Ad5 genome E2B E2A E4 Transgene 1st generation E1(+/-)-deleted Immunogenic response Transgene 2nd generation E1(+/-E3)+E2/4-deleted E1A Transgene I Transgene II High-capacity vector E1A Non-coding stuffer DNA gutless Recombinant Adenovirus propagated in the cell line expressing E1 region Advantage of Adenovirus: Relatively simple genetics Can accommodate large insert allows controllable expression High viral titer Infect non-dividing cell Disadvantage of Adenovirus : Non-integrative Transient expression Immunogenic response observed Adeno Associated vector ITR 145 bp rep cap ITR 145 bp r AAV production Transcription unit ITR ITR rep cap Helper adenovirus 293 cell Mixed helper /r AAv Heat 56oC CsCl2 gradient centrifugation Recombinant AAV Advantage of Adeno associated virus: Parvoviridae family Non human disease associate Integrate stably into chromosome 19( site specific integration) Transduce mitotic and post mitotic Disadvantage of Adeno associated virus: Limited host range High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors https://www.jove.com/details.php?id=2538 Gene therapy for bone regeneration Scheller & Krebsbach etal , J Dent Res 88(7) 2009