Gene Transfer. Comparison of different viral vectors Viral vectortitersmanupilation of...
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Transcript of Gene Transfer. Comparison of different viral vectors Viral vectortitersmanupilation of...
Gene Transfer
Comparison of different viral vectors
Viral vector titers manupilation of immunogenicity infecting of
tropism non-dividing cells
Adenovirus 1011 terrific very high yes
Retrovirus 107 good low only lentivirus
Herpesvirus 107 not so good low yes
AAV 107 not so good low yes
2nd 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)