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DNA Transfection to Mammalian Cells Three essential tools form the basis for studying the function of mammalian genes: 1.Isolate a gene by DNA cloning 2.manipulate the sequence of a gene in the test tube 3. The technique should be able to return the altered gene to cells to determine the function Extract DNA with restriction endonuclease or RNA and prepare cDNA Grow up cells transfected cells in selective medium, and assay for expression Trasfection into recipient cells with lipofection, calcium phosphate or electroporation Incorporate into plasmid with selectable marker Clone in bacteria in selective condition The first methods used for DNA transfection 1. DEAE( Diethylamine ethyl) positively charged enter cells y endocytosis 2. Calcium Phosphate divalent cations promote DNA entry in bacterial cells Exogenous DNA is Transiently or Stably Expressed 1. Transient Transfection DNA expressed immediately after transfection Assay by reporter i.e. C.A.T. :chloramphenical acetyl transferase RNA transcription i.e. northern blotting 2. Stable Ttransfection Clone selected by G418 ( geneticin) or hygromycin may be used to obtain high protein gene amplification expression by Dominant selectable markers Used in transfection experiments 1.Aminoglycoside phosphotransferase(APH) G418( inhibit protein synthesis.) APH inactivate G418 2.Dihydrofolate reductase (DHFR):Mtx-resistant Methorexate( inhibit DHFR) variant DHFR resist to Mtx 3.Hygromycin-B-Phoshotransferase (HPH) Hygromycin-B( inhibit protein synthesis) HPH inactivate hygromycin B 4.Thymidine kinase(TK) Aminopeterine( inhibits de novo purine and thymidylate) TK synthesize thymidylate 5. Xanthine-guanine phosphoribosyltransferase(XGPRT) mycophenolic acid( inhibits de novo GMP synthesis) XGPRT synthesize GMP from xanthine 6. Adenosine deaminase(ADA) 9--xylofuranosyl adenine(Xyl-A; damages DNA) ADA inactivate Xyl-A Specific methods used For Transfection 1. Electroporation a brief change of electric pulse discharges across the electrode, transiently open holes in cells 2. Liposomediated gene transfer liposome fuse directly with cell membrane and delivers DNA into cells Virus Vectors 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 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) Developing baculovirus-insect cell expression system for humanized recombinant glycoprotein 4. Retrovirus 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 5‘LTR gag casset gagORF antibioticsR 3‘LTR polORF envORF Genome 7-10kb contain gag, pro, pol, env : encode structural capsid proteins, viral protease, integrase, and viral reverse transcriptase, enveloped glycoproteins Advantages of retrovirus vector Stably traduce dividing cells Long term transgene expression Disadvantage of retrovirus vector Random insertion into host cell and causes oncogenic activation or tumour-suppressor gene inactivation Limited insert capacity( 8kb) Low titer Inactivatoion by human complement Inability to transduce nondividing cells Retrovirus life cycle Adeno virus Non-envelope d.s DNA virus Genome :36kB ITR Early E1A Late ITR CAR receptor pH dependent release of virus particle Immunogenic response gutless Recombinant Adenovirus propagated in the cell line expressing E1 region Adeno Associated vector Parvoviridae family Non human disease associate Integrate stably into chromosome 19 Transduce mitotic and post mitotic cells 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