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Viral Vectors

Viral Vectors. May 8. Viral vectors. Herpes simplex virus amplicons Adenovirus vectors Lentivirus vectors. Herpes simplex virus amplicons. HSV replicative cycle. cell-cell spread. inclusion body. suppression of class I MHC. HSV amplicons. cloning site. defective virus. promoter.

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Viral Vectors

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  1. Viral Vectors May 8

  2. Viral vectors • Herpes simplex virus amplicons • Adenovirus vectors • Lentivirus vectors

  3. Herpes simplex virus amplicons

  4. HSV replicative cycle cell-cell spread inclusion body suppression of class I MHC

  5. HSV amplicons cloning site defective virus promoter pA pack cosmids HSV genes HSV-ori bacterial plasmid seq BACS HSV genes linear plasmid joined end to end 150kb packaged into virus particle

  6. HSV ampliconsadvantages: • large packaging capacity • for small inserts - large gene dose • extra-chromosomal • wide host range • no viral sequences except ori and pkg

  7. HSV ampliconsdisadvantages • often low virus titres • short-term expression due to silencing

  8. Improving expression by amplicons Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin Masataka Suzuki, Kazue Kasai, and Yoshinaga Saeki, J. Virol 80: 3293, April 2006

  9. Adenovirus vectors • gene of interest part of defective virus genome • exrta-chromosomal

  10. Clontech Adeno-X expression system

  11. Lentivirus expression systems • contain no genes for viral structural genes • wide host range if VSV-G used • integrate into host chromosome (dividing or stationary cells)

  12. A retrovirus virion

  13. The viral genome (oncornaviruses) Long terminal repeat RNA dependant DNA polymerase LTR LTR GAG POL ENV Envelope proteins (type-specific antigens) Group specific antigens (nucleocapsid) All three genes - GAG, POL, ENV - required for replication

  14. Replicative cycle

  15. Directional cloning into pLenti

  16. Invitrogen Lentivirus vector system clone gene of interest lentivirus virions with gene of interest cells into target cells plasmids expressing: HIV Gag and Pol HIV Rev VSV G stably expressing cells (blasticidin) transient expression

  17. Primers for amplifying ZF sequence at 5’ end of ZF FLAG 5’ ATG GAC TAC AAA GAC GAT GAC GAC AAG CAA TTCATG GAG GAG GAG GCG ATC GCC AGC CTC CCG GGG GAA GAG… Upstream primers FLAG+ 5’ CACC ATG GAC TAC AAA GAC GAT G FLAG- 5’ CACC ATG GAG GAG GAG GCG ATC G

  18. Primers for amplifying ZF Sequence of 3’ end of cds of hZhangfei 5’ GCG TCG TCT TCT CTT AAA ATG TAG 3’ CGC AGC AGA AGA GAA TTT TAC ATC Down stream primer: 5’ CAT TTT AAG AGA AGA CGA CGC 3’ Stop codon removed and sequence in phase with SV5 epitope No CACC at 5’ end

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