1. Molecular methods of cell culture II Expression of mammalian expression vector in cell culture

2.  -Galactosidase Reporter system  Hydrolaze enzyme catalyze hydrolysis of  -galactoside to monosaccharide  Contain 1023 a.a.

3. Mammalian  -Galactosidase Reporter system

4. Expression of  -Galactosidase in WI-380 cells

5. 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 TRE: Tetracyclin( Doxicyclin) response element Tet on Inducible system Induction by Doxicyclin

6. Expression of Fluorescent protein in cell culture Green fluorescent protein ( GFP) 238 a.a or Red Fluorescent protein Cloned from jelly fish( Aequorea victoria)

7. Red Fluorescent protein fusion protein to the multiple cloning site

8. HEL: Human Embryonic Lung GFP DAPI

9. RFP DAPI HEL: Human Embryonic Lung

11. Promoter reporter system Luciferase assay Luciferase gene Luciferase MCS Multiple cloning site SV40

12. 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

13. 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

14. DNA :polymer complex DNA :polymer complex DNA :polymer complex endosome Episomal or integration with selection Transcription Entry of DNA-Polymer Complex

15. Virus vector  Gene delivery in cell culture system  Deliver vector for gene therapy  Recombinant protein production in industry

16. Adenovirus( Tumors, Haemopoietic cells Retrovirus( t umor,stem,h aemopoitic cells Herpes simplex virus(CNS, PNS,muscle, Haemopoiet ic, stem cells AAV( liver, muscle, retinal Polymer coated adenovirus ( tumors) Liposome encapsula te alpha virus Lentivirus( CNS, liver,muscl e Alpha virus ( tumors) Clinical application of viral vector TRENDS in Biotechnology Vol.21 No.3: 119 2003

17. 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

18. Virus Vectors retrovirus

19. Recombinant cell lines

20. 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

21. SV-40 T ag integration T ag protein allows replication of plasmid

22. 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

23. Baculovirus  Foreign gene maybe coexpressed with structural gene ( structural protein expresses when infection occurs) replacement of coat protein gene with gene of interest

24. Baculovirus Replace coat protein gene with gene of interest

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

26. Baculovirus-insect cell expression system

27. 4. Retrovirus Recombinant retroviruses used in gene delivery: 1.γ-retroviruses 2. Lentiviruses 3. spumaviruses

28. Retrovirus life cycle

29. U3RU5 PBS  gagpropol env U3RU5 PPT att 5’LTR 3’LTR 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 Genome 7-10kb contain gag, pro, pol, env : encode structural capsid proteins, viral protease, integrase, and viral reverse transcriptase, enveloped glycoproteins

30.  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 U3RU5 PBS  cPPT Transgene P att 5’LTR U3RU5 att 3’LTR amp r U3RU5 PBS  gagpropol env U3RU5 PPT att 5’LTR 3’LTR

31. RSVRU5 PBS  cPPT Transgene att 5’LTR U3RU5 att 3’LTR CI,CR,WPREs/MAR,amp r P Retroviral vector development for increased efficiency and targeting Structure of an enhanced self-inactivating retroviral gene therapy vector  substitution of the 5′LTR U3 component by RSV.  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 U3RU5 PBS  gagpropol env U3RU5 PPT att 5’LTR 3’LTR

32. Function of 3rd generation MLV packing cell line Therapeutic gene Stable integration

33. 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

34. 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

35. Lentivirus human immunodeficiency virus (HIV) simian immunodeficiency( SIV) non-primate- equine infectious anaemia virus (EIAV) feline immunodeficiency virus (FIV) Accessory and Regulatory genes  oncogenesis (tat), apoptosis (vpr), MHC downregulation, (nef ) differentiation (vpu)  Biologically active and can cause detrimental effects on cells 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

36. Lenti virus vectors lacking tat, vpr, nef vpu TU, transducing units; p, promoter; env, envelope; WPRE, Woodchuck hepatitis post- transcriptional regulatory element; cPPT, central polypurine tract; CMVp, cytomegalovirus promoter

37. J Gene Med 2004; 6: S83–S94. 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

38. http://www.jove.com/details.php?id=2307 Retroviral Transduction of T-cell Receptors in Mouse T-cells

39. Adeno virus Largest non-enveloped virus and contain linear, double-stranded Genome :36kB Early genes : function as regulatory proteins for viral replication Late genes : encode structural proteins for new virus assembly ITREarly Late E1AITR E1A gene: a trans-acting transcriptional regulatory factor that is required for early gene activation

41. CAR receptor pH dependent release of virus particle

42. Group C: most common in nature and in adenoviral gene therapy are human serotypes 2 and 5 Adenovirus Serotype

43. E1AE1BMLP L1L2L3L4 E3 L5 Ad5 genome E2BE2AE4 Immunogenic response Transgene 1st generation E1(+/-)-deleted Transgene 2nd generation E1(+/-E3)+E2/4-deleted E1A gutless Transgene I High-capacity vector E1A Transgene II Genome type of Adenovirus and different types of Ad5 derived vector Non-coding stuffer DNA

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

46. Advantage of Adenovirus:  Relatively simple genetics  Can accommodate large insert allows controllable expression  High viral titer  Infect non-dividing cell

47. Disadvantage of Adenovirus :  Non-integrative  Transient expression  Immunogenic response observed

48. repcap ITR 145 bp Adeno Associated vector

49. r AAV production Transcription unit Helper adenovirus rep cap 293 cell Mixed helper /r AAv ITR Heat 56 o C CsCl 2 gradient centrifugation Recombinant AAV

50. 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

51. https://www.jove.com/details.php?id=2538 High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

52. Gene therapy for bone regeneration Scheller & Krebsbach etal, J Dent Res 88(7) 2009