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 inclusion body cell-cell spread suppression of class I MHC

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

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

7. HSV amplicons disadvantages 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) LTR GAGPOLENV Long terminal repeat Group specific antigens (nucleocapsid) RNA dependant DNA polymerase Envelope proteins (type-specific antigens) All three genes - GAG, POL, ENV - required for replication

14. Replicative cycle

16. Directional cloning into pLenti

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

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

19. 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