Download May 8: Viral vectors

Viral Vectors
May 8
Viral vectors
• Herpes simplex virus amplicons
• Adenovirus vectors
• Lentivirus vectors
Herpes simplex virus
amplicons
HSV replicative cycle
vhs
gC
gD
gB
inclusion body
viral DNA
IE genes
E genes
DNA replication
L genes
tif
suppression of
class I MHC
cell-cell
spread
HSV amplicons
cloning site
promoter
pA
defective
virus
pack
linear plasmid joined end to end
150kb packaged into virus particle
HSV-ori
bacterial plasmid seq
cosmids
HSV genes
BACS
HSV genes
HSV amplicons
advantages:
•
•
•
•
•
large packaging capacity
for small inserts - large gene dose
extra-chromosomal
wide host range
no viral sequences except ori and pkg
HSV amplicons
disadvantages
• often low virus titres
• short-term expression due to silencing
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
Adenovirus vectors
• gene of interest part
of defective virus
genome
• exrta-chromosomal
Clontech
Adeno-X
expression
system
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)
A retrovirus virion
envelope
(ENV)
p15e
gp70
RNA dep. DNA pol
(POL)
nucleocapsid
p27 (GAG)
genome (2 copies)
The viral genome
Long terminal(oncornaviruses)
repeat
LTR
RNA dependant DNA
polymerase
GAG
Group specific antigens
(nucleocapsid)
POL
ENV
LTR
Envelope proteins
(type-specific antigens)
All three genes - GAG, POL, ENV - required for replication
Replicative cycle
receptor
uncoating
RNA
viral dsDNA integrated
into host genome
blocked
receptors
ds DNA
mRNA
proteins
pro-virus
genomic RNA
assembly
Directional cloning into pLenti
Invitrogen Lentivirus vector
clone gene of interest system
lentivirus virions
with gene of
interest
cells
plasmids expressing:
HIV Gag and Pol
HIV Rev
VSV G
into target cells
stably
expressing
cells
(blasticidin)
transient
expression
Primers for amplifying ZF
sequence at 5’ end of ZF
FLAG
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…
Upstream primers
FLAG+
5’ CACC ATG GAC TAC AAA GAC GAT G
FLAG5’ CACC ATG GAG GAG GAG GCG ATC G
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