lentiviral vectors: design, production, and titration · lentiviral vectors: design, production,...
TRANSCRIPT
Lentiviral Vectors:design, production,
and titration
ONPRC Lentiviral Vector CoreMolecular and Cellular Biology Core
Greg Dissen
Gag LTRLTR Pro
U
PolEnvVif
Nef
R
RRE
RevTat
HIV provirus
SDψ
∆∆∆
2nd and 3rd generation viral vectors
1. Viral backbone was stripped to allow room for transgenes
2. Development of the Self-Inactivating (SIN) vector
Lentiviral Vector System
2. Modification of 3’LTR “Self Inactivating”
U3 R U5-456 +1 +60 +181
AP-1
NF-AT?
USFEts
TCF-1α
NF-κBNF-ATc
SP1
TATA
-418EcoRV
-18PvuII
∆U3 R U5pHR’ SIN-18
400 bp Deletion
Wild-Type HIV 3’ LTR
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System (3rd generation)
∆U3 R U5DU3 R U5
SDΨ
∆U3 R U5∆U3 R U5
Integration into theHost Genome
Self InactivationNo LTR promoter interference
Gag LTRLTR Pro
U
PolEnvVif
Nef
R
RRE
RevTat
HIV provirus
SDψ
Lentiviral Vector Systems
∆U3 R U5DU3 R U5
∆
∆U3 R U5DU3 R U5
2nd Generation vector
3rd Generation vector
Requires Tat for production
Tat is not required
SIN
SIN
Constitutive promoterRSV or CMV
RSV
Gag LTRLTR Pro
U
PolEnvVif
Nef
R
RRE
TatRev
HIV provirus
SD ψ
Lentiviral Vector Generations
1st Generation:
2nd Generation:
3rd Generation:
HIV provirus:
pMDLgpRRE or pLP1
pRSV-Rev or pLP2
HIV-1 core proteinsEnzymes and Accessory factorsFrom separate plasmidAnd env plasmid
pLV
pMD.G+
+
+pLV
pMD.G
pLV
pMD.G
Packaging reducedgag, pol, tat, revAnd env plasmid
Requirement for tatEliminated, revMoved to separate plasmid
Packaging plasmids
4th generation?
Clontech
5’LTR cPPT wPRE SIN3’LTR
centralpolypurinetract
woodchuckhepatitis virusposttranscriptionalregulatoryelement
RRE
Revresponsiveelement
Lentiviral Vector Systems
pHR’ SIN-18
∆U3 R U5DU3 R U5
3rd GenConstitutivePromoter:RSVCMV
SDΨ
PackagingRegion
SpliceDonorSite
Both cPPT and wPRE increaseTransduction efficiency and transgene expression
Rev is essential for viral replicationBinds mRNAs removing them from splicesome = full-length and partially spliced
8 KB
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector Systems
∆U3 R U5DU3 R U5
SDΨ
RNA Polymerase II
Constitutive:CMVSV40hEFpPGK
Tissue Specific
Reporter
Reporter Vector
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System (3rd generation)
∆U3 R U5DU3 R U5
SDΨ
New components:
∆U3 R U5DU3 R U5
IRES
Allows the production of two proteins from one mRNA. A Bicistronic RNA.
Internal Ribosome Entry Site:
∆U3 R U5DU3 R U5
2nd
promoter
Allows the production of two mRNAs from one vector.
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System (3rd generation)
∆U3 R U5DU3 R U5
SDΨ
New components:
∆U3 R U5DU3 R U5
IRESMCS
Multiple Cloning Site for transgenes to be expressed:
∆U3 R U5DU3 R U5
IRES
Transgene
2nd
promoter
2nd
promoter
Lentiviral Vector System (3rd generation)
∆U3 R U5DU3 R U5
IRES
Transgene
Insertion of a heterologous Intron
∆U3 R U5DU3 R U5Transgene
Intron
A heterologous intron had been found to increase expression of transgenesin transgenic mice.
2nd
promoter
Rat insulin II intron A
Lentiviral Vector System (3rd generation)
∆U3 R U5DU3 R U5
IRES
NGF
∆U3 R U5DU3 R U5
IRES
NGF
Intron
200
150
100
50
0IntronNo IntronLV
ng/m
l med
ium
CMV
Small peptideLigand is Produced andExpression isEnhancedFrom vector ContainingHeterologousIntron
Lentiviral Vector System (3rd generation)IRES
∆U3 R U5DU3 R U5
Jag
hEFp
eGFP∆U3 R U5DU3 R U5
IntronCMVp
∆U3 R U5DU3 R U5
∆U3 R U5DU3 R U5
∆U3 R U5DU3 R U5
Jag
GAPDH
GFP
1
2
3
4
5
1 2 3 4 5
140 kD
42 kD
27 kD
Lentiviral Vector System (3rd generation)IRES
∆U3 R U5DU3 R U5
Jag
hEFp
eGFP∆U3 R U5DU3 R U5
IntronCMVp
∆U3 R U5DU3 R U5
∆U3 R U5DU3 R U5
∆U3 R U5DU3 R U5
Jag
GAPDH
GFP
1
2
3
4
5
1 2 3 4 5
GAPDH (36kDa)
eGFP (27kDa)
FXYD (14kDa)
polybrene LV FIG LV GIF
1:10 1:20 1:30 1:10 1:20 1:30
Infection of Hib5 (6 Well plate, 200,000 cells/well)
LV FIG (1:30) LV GIF (1:30)
FXYD1 EGFP
IRES
FXYD1EGFP
IRESCMV CMV
miR30=Retinoblastoma (Rb)Targeting sequence:AGCAGTTCGATATCTACTGAAA
Stegmeier, 2005
pPRIMEPotent RNA Interferenceusing MicroRNA Expression
pPRIME-CMV-GFPmiR30-shRNA
Pol II driven shRNAwas more active than the Pol IIIconstruct
pPRIME-TET-GFPCMV-dsREDCMV-NeoT-REX-GFP
cPPT Xho I EcoRI
CMV ∆U3 R U5DU3 R U5 3’miR305’miR30GFPCMV CM R WRERRE
Lentiviral Vector System: Gene Suppression
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System: Gene Suppression
∆U3 R U5DU3 R U5
SDΨ
New components:
∆U3 R U5DU3 R U5
U6 promoterT3
U6-siRNA MCS Cassette
* *
486 bp
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System: Gene Suppression
∆U3 R U5DU3 R U5
SDΨ
New components:
∆U3 R U5DU3 R U5
U6 promoterT3
U6-siRNA MCS Cassette
* *
486 bp
∆U3
U6-siRNA
Virus Production
1. Cells: human Embryonic Kidney 293T/17Cells have been transformed with temperature sensitive large T antigen Strain was selected specifically for its high transfectability
2. Cells are grown in antibiotic free conditions DMEM (1.5 g/l Na Bicarbonate), 4.5 g/l Glucose, Defined fetal bovine serum, 10% CO2 Advantage to antibiotic free medium = immediately know when there is a problem/contamination
3. Cells are plated to achieve 70 confluency in 10 cm dishes that havebeen coated with poly-L-lysine (6 to 11 x 106 cells/dish)
Day 1
pLV7,438 bp
SIN3’ LTRWPRE
eGFP
hCMV-PcPPT5’LTR RRE
pMD.G6,010 bp
Poly A
VSV G
hβ GlobinIVS2
hCMV-P
pMDLgpRRE8,895 bp
Pol
GAG
hCMV-P
RREPoly A
pRSV-Rev4,174 bp
Poly A
RSV
REV
PackagingTransgene Envelope
Lentiviral Vector System
TransfectionpLV
pMDLpg.RRE pRSV-RevpMD.G
CaPO4 CaPO4
pLV7,438 bp
SIN3’ LTRWPRE
eGFP
hCMV-PcPPT5’LTR RRE
pMD.G6,010 bp
Poly A
VSV G
hβ GlobinIVS2
hCMV-P
pMDLgpRRE8,895 bp
Pol
GAG
hCMV-P
RREPoly A
pRSV-Rev4,174 bp
Poly A
RSV
REV
PackagingTransgene Envelope
Lentiviral Vector System
TransfectionpLV
pMDLpg.RRE pRSV-RevpMD.G
Transfected 293T cellsCaPO4 CaPO4
M1
M1
99.18%
99.8%
HEPES - Transfection
BES - Transfection
M1
0.29%Control
Transfection
Infection
pLVpMDLpg.RRE pRSV-Rev
pMD.G
Conditioned Medium
Transfected 293T cells
Infected 293T cellsFACs detects Infected cells, Expressed asPercentage of total
FACs Titer:
Virus Production
Titer Analysis Possibilities:FACS for gene expression product:
Real-Time PCR for integrated viral DNA in host genome
Reverse transcription Real-Time PCR for viral RNA
Dependent on Promoter activityConstitutive promoter = useful Titers that predict infection rateTissue specific promoters might not give useful titers
Dependent on infection and integration into the host genomeReal-Time PCR Titers predict infection rate
Dependent only on the presence of the viral RNADoes not predict infection rate of the viral particles
Acknowledgements
Molecular and Cellular Biology CoreOregon National Primate Research Center
Eliot Spindel, MD., [email protected]
Yibing Jia, M.S.DNA Sequencing, Realtime PCR & [email protected]
CoreyAyne Singleton, M.S.Cell culture, Genomic DNA preparation, Lentivirus [email protected]
Greg Dissen, Ph.D.Director, Lentiviral [email protected]
0
10
20
30
40
50
60
70
0 0.5 1 1.5 20
10
20
30
40
50
60
70
0 0.5 1 1.5 20
10
20
30
40
50
60
70
0 0.5 1 1.5 2
CMVshort GnRHlong GnRH
Fluorescence in 293-T embryonic kidney cells
viral prep (µl)
% fl
uore
scen
ce
Noriega
0
10
20
30
40
50
60
0 50 100 150 200 250
CMVshort GnRHlong GnRH
viral prep (µl)
Fluorescence in GT1-7 neuronal cells%
fluo
resc
ence
Noriega
Replication Competent Lentivirus (RCL)
Gag LTRLTR Pro
U
PolEnvVif
Nef
R
RRE
TatRev
HIV provirus
SD ψ
Wild-Type Virus
3rd Generation Lentiviral Vector
+Replication competent LTRGag,pol, rev, env, tat
Source:Carry over from packaging orEnvelope plasmidsOrEndogenous viruses
Replication Competent Lentivirus (RCL)Protocol:
1. Infect 1 million SupT-1 cells with 5 million viral TUs
2. Pass the cells 3 times over 2-3 weeks
3. Test the medium for p24 protein with ELISA kit (commercial)
+ StdTest
Preps