1. Gene Transfer

2. Comparison of different viral vectors Viral vectortitersmanupilation of immunogenicityinfecting of tropism non-dividing cells Adenovirus10 11 terrificvery highyes Retrovirus10 7 goodlowonly lentivirus Herpesvirus10 7 not so goodlowyes AAV10 7 not so goodlowyes

5. 2 nd generation lentiviral plasmids

6. 3rd generation lentiviral plasmids

8. Retroviral vector Moloney murine leukemia virus (MuLV) Generation of replication defective retroviral vector – Transfer plasmid vector: Gene of interest Long terminal repeats(LTR): promoter, polyA, integration, replication and reverse transcription Primer binding site (PBS) (origin of replication) RNA packaging signal Poly purine tract (important for replication) – Packaging vector Cell line stably transfected with plasmid constructs containing Gag/pol and Env

9. Generation of retroviral vector

10. Pseudotyped retroviral vector

12. Characteristics of retroviral vector Advantages – Integration: permanent expression – Pseudotyped virus Disadvantages – Only infecting dividing cells – Insertional mutagenesis (tumor formation) Activate oncogenes Inhibit tumor suppressor genes

13. Genome Editing Zinc finger Nucleases TALENs (transcription activator-like effectors (TALEs) nucleases) Zinc finger Nickases

16. Xanthomonas, producer of TALE proteins

19. NHEJ

20. HDR (homology-directed repair)