1. 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

2. Extract DNA or RNA and prepare cDNA with restriction endonuclease Incorporate into plasmid with selectable marker Clone in bacteria in selective condition Trasfection into recipient cells with lipofection, calcium phosphate or electroporation Grow up cells transfected cells in selective medium, and assay for expression

3. 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

4. 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

5. 2. Stable Ttransfection  Clone selected by G418 ( geneticin) or hygromycin  may be used to obtain high protein expression by gene amplification

6. 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

9. 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

10. 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

11. 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

14. Virus Vectors

15. 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

17. 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

18. Baculovirus foreign gene maybe coexpressed with structural gene ( structural protein expresses when infection occurs)

20. Developing baculovirus-insect cell expression system for humanized recombinant glycoprotein

22. 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

23. Genome 7-10kb contain gag, pro, pol, env : encode structural capsid proteins, viral protease, integrase, and viral reverse transcriptase, enveloped glycoproteins gagcassetantibiotics R 3‘LTR 5‘LTR gagORFpolORF envORF 

24. 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

25. Retrovirus life cycle

26. Adeno virus Non-envelope d.s DNA virus Genome :36kB EarlyLate ITR E1A ITR

28. CAR receptor pH dependent release of virus particle

30. Immunogenic response gutless

32. Recombinant Adenovirus propagated in the cell line expressing E1 region

33. Adeno Associated vector  Parvoviridae family  Non human disease associate  Integrate stably into chromosome 19  Transduce mitotic and post mitotic cells repcap ITR 145 bp

34. Transcription unit Helper adenovirus ITR rep cap 293 cell Mixed helper /r AAv r AAV production

35. Heat 56 o C CsCl 2 gradient centrifugation Recombinant AAV