The lentiviral expression system we use is derived from Invitrogen’s multi-site Gateway Expression system. This is a powerful system allowing individuals to select the specific:
- 1) promoter
- 2) gene of interest, and
- 3) lentiviral backbone
These plasmids can then be recombined to create a 3rd generation lentiviral transfer vector. To create functional virus, this vector is then co-transfected into HEK293FT packaging cells with the packaging plasmids: pLP1 (gag/pol), pLP2(Rev), and pLP-VSVG (vesicular stomatitus virus glycoprotein). These lentiviral virions are capable of infecting a wide-range of cell types.
To get started, examine: Invitrogen’s user’s manual
Invitrogen’s system came with UbC promoter and a Dest vector that conferred Blasticidin resistance. Over the years we have modified and expanded this system to form a very powerful toolbox of plasmids and vectors.
PROMOTERS
GENES
pLENTI Backbones
pENTR 5′- UbC pro
pENTR1A-CRE
pLenti6/R4R2/V5-DEST (Blasticidin)
pENTR 5′- CMV pro
pENTR1A-p16-IRES-EGFP
pLenti6/R4R2/V5-DEST (Neo)
pENTR 5′- EF1alpha pro
pENTR1A-SV40 small + Large T antigen
pLenti6/R4R2/V5-DEST (Cerulean)
pENTR 5′-pENTR 5′- HSV-TK pro
pENTR1A-GSE22
pLenti6/R4R2/V5-DEST (EGFP)
pENTR 5′- TRE-TIGHT
pENTR1A- HPV16 E7
pLenti6/R4R2/V5-DEST (ZsGreen)
pENTR 5′- Keratin 19 pro
pENTR1A- GFP-53BP1
pLenti6/R4R2/V5-DEST (Citrine)
pENTR 5′- Keratin 14 pro
pENTR D-HPV16 E6/E7
Lenti6/R4R2/V5-DEST (mCherry)
pENTR 5′- 15-ERE pro (estrogen response)
pENTR D-HPV16 E6
pLenti6/R4R2/V5-DEST (DsRed Express)
pENTR 5′- 25-ERE pro (estrogen response)
pENTR221-hRasV12
pLenti6/R4R2/V5-DEST (Luc2)
pENTR 5′- TRE-TIGHT (reverse orientation) (reverse orientation)
pENTR D-HER2 V659 (CA)
pLenti6/R4R2/V5-DEST (hRas V12)
pENTR D-Cerulean
pLenti6/R4R2/V5-DEST (H2b-GFP)
pENTR D-ZsGreen
pLenti6/R4R2/V5-DEST (rtTA3) TET-ON Advanced
pENTR D-ZsGreen-DR
pGEMTez-R4-cm-ccdb-R2 DEST
pENTR D-EGFP
pENTR D-H2B-EGFP
pENTR D-H2B-mCherry
pENTR D-H2B-Orange
pENTR D-H2B-Venus
pENTR D-H2B-CFP
pENTR D-ZsYellow
pENTR D-yPET
pENTR D-DsRED-Express
pENTR D-DsRED-Express-DR
pENTR D-mCherry
pENTR D-mCherry-NLS
pENTR D-Brainbow 1.0
pENTR D-NEO
pENTR D-PURO
pENTR D-rtTA3 Tet-ON Advanced
pENTR D-Thymidine Kinase
pENTR D-Luc2 Luciferase
pENTR D-EGFP 5’miR|XhoI/EcoRI|3’miR
pENTR D-5’miR|XhoI/EcoRI|3’miR
pENTR GUS (ß-glucuronidase) reporter
pENTR D-H2B-HcRed
Lentivirus can be created with VSVG or other glycoproteins I have:
| PACKAGING |
VSV-G |
| GP64 |
| Mokola |
| Rabies |
| LCMV-WE |
| LCMV |
| Ampho |
| 10A1 |
| Ebola |
| Ebola-dZ |
| Sindbis |
| ZZ-Sindbis |
Protocols:
TOPO Cloning entry vectors
LR Reaction spreadsheet
Calculating the amount of DNA for LR reaction
Packaging Lentivirus
Link to these to Addgene once they are deposited.
Cite refs for glycoproteins
