Gene Transfer. Comparison of different viral vectors Viral vectortitersmanupilation of immunogenicityinfecting of tropism non-dividing cells Adenovirus10.

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Presentation transcript:

Gene Transfer

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

2 nd generation lentiviral plasmids

3rd generation lentiviral plasmids

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

Generation of retroviral vector

Pseudotyped retroviral vector

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

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

Xanthomonas, producer of TALE proteins

NHEJ

HDR (homology-directed repair)

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