Lentiviral Expression System

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.

Picture1

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

 

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