viral vectors
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Viral Vectors
May 8
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Viral vectors
• Herpes simplex virus amplicons• Adenovirus vectors• Lentivirus vectors
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Herpes simplex virus amplicons
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HSV replicative cycle
IE genesE genes
L genesDNA replication
viral DNA
gB
gC
gD
vhs
tif
inclusion body cell-cellspreadsuppression of
class I MHC
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HSV amplicons
promoter pA pack
HSV-ori
bacterial plasmid seq
cloning site defectivevirus
cosmidsHSV genes
BACSHSV geneslinear plasmid joined end to end
150kb packaged into virus particle
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HSV ampliconsadvantages:
• large packaging capacity• for small inserts - large gene dose• extra-chromosomal• wide host range• no viral sequences except ori and pkg
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HSV ampliconsdisadvantages
• often low virus titres• short-term expression due to silencing
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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
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Adenovirus vectors
• gene of interest part of defective virus genome
• exrta-chromosomal
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Clontech Adeno-X expression system
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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)
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A retrovirus virionenvelope (ENV)
nucleocapsidp27 (GAG)
genome (2 copies)
RNA dep. DNA pol(POL)
p15e
gp70
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The viral genome (oncornaviruses)
LTR LTRGAG POL ENV
Long terminalrepeat
Group specific antigens(nucleocapsid)
RNA dependant DNApolymerase
Envelope proteins(type-specific antigens)
All three genes - GAG, POL, ENV - required for replication
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Replicative cyclereceptor
uncoating
RNA
ds DNA
proteinsmRNA
viral dsDNA integratedinto host genome
genomic RNA
blockedreceptors
assemblypro-virus
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Directional cloning into pLenti
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Invitrogen Lentivirus vector systemclone gene of interest
plasmids expressing:HIV Gag and PolHIV RevVSV G
cells
lentivirus virions with gene of interest
into target cells
stably expressingcells(blasticidin)
transient expression
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Primers for amplifying ZFsequence 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 primersFLAG+5’ CACC ATG GAC TAC AAA GAC GAT G
FLAG-5’ CACC ATG GAG GAG GAG GCG ATC G
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Primers for amplifying ZFSequence 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 epitopeNo CACC at 5’ end