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.
[ezcol_1third]PROMOTERS[/ezcol_1third] [ezcol_1third]GENES[/ezcol_1third] [ezcol_1third_end] pLENTI Backbones [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- UbC pro [/ezcol_1third] [ezcol_1third] pENTR1A-CRE [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (Blasticidin) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- CMV pro [/ezcol_1third] [ezcol_1third] pENTR1A-p16-IRES-EGFP [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (Neo) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- EF1alpha pro [/ezcol_1third] [ezcol_1third] pENTR1A-SV40 small + Large T antigen [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (Cerulean) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′-pENTR 5′- HSV-TK pro[/ezcol_1third] [ezcol_1third] pENTR1A-GSE22 [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (EGFP) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- TRE-TIGHT [/ezcol_1third] [ezcol_1third] pENTR1A- HPV16 E7 [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (ZsGreen) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- Keratin 19 pro [/ezcol_1third] [ezcol_1third] pENTR1A- GFP-53BP1 [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (Citrine) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- Keratin 14 pro[/ezcol_1third] [ezcol_1third] pENTR D-HPV16 E6/E7 [/ezcol_1third] [ezcol_1third_end] Lenti6/R4R2/V5-DEST (mCherry) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- 15-ERE pro (estrogen response) [/ezcol_1third] [ezcol_1third] pENTR D-HPV16 E6 [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (DsRed Express) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- 25-ERE pro (estrogen response) [/ezcol_1third] [ezcol_1third] pENTR221-hRasV12 [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (Luc2) [/ezcol_1third_end]
[ezcol_1third] pENTR 5′- TRE-TIGHT (reverse orientation) (reverse orientation)[/ezcol_1third] [ezcol_1third] pENTR D-HER2 V659 (CA) [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (hRas V12) [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-Cerulean [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (H2b-GFP) [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-ZsGreen [/ezcol_1third] [ezcol_1third_end] pLenti6/R4R2/V5-DEST (rtTA3) TET-ON Advanced [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-ZsGreen-DR [/ezcol_1third] [ezcol_1third_end] pGEMTez-R4-cm-ccdb-R2 DEST [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-EGFP [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-EGFP [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-mCherry [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-Orange [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-Venus [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-CFP [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-ZsYellow [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-yPET [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-DsRED-Express [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-DsRED-Express-DR [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-mCherry [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-mCherry-NLS [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-Brainbow 1.0 [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-NEO [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-PURO [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-rtTA3 Tet-ON Advanced [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-Thymidine Kinase [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-Luc2 Luciferase [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-EGFP 5’miR|XhoI/EcoRI|3’miR [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-5’miR|XhoI/EcoRI|3’miR [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR GUS (ß-glucuronidase) reporter [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] pENTR D-H2B-HcRed [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
[ezcol_1third] [/ezcol_1third] [ezcol_1third] [/ezcol_1third] [ezcol_1third_end] [/ezcol_1third_end]
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:
Calculating the amount of DNA for LR reaction
Link to these to Addgene once they are deposited.
Cite refs for glycoproteins