TRC I Vector - pLKO.1-puro Vector and Use
Features of the pLKO.1-Puro vector allow for transient or stable transfection of the shRNA as well as production of lentiviral particles.1 Stable gene silencing is selected using the puromycin selectable marker while self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK 293T) by co-transfection with compatible packaging plasmids.2,3 Unlike adenovirus or murine-based MMLV or MSCV retroviral systems, lentiviral-based particles permit efficient infection and integration of the specific shRNA construct into differentiated and non-dividing cells, such as neurons and dendritic cells, overcoming low transfection and integration difficulties when using these cell lines. Compared to siRNA and other vector-based systems, pLKO.1 provides solutions for long-term knockdown and phenotypic observation, transduction of difficult or sensitive cell lines (non-dividing cells or primary cells), and is an economical renewable resource.
pLKO.1 puro sequence (8 Kb txt file)
Vector Map
pLKO.1-puro vector description and features
| Name |
Description |
| cppt |
Central polypurine tract |
| hPGK |
Human phosphoglycerate kinase eukaryotic promoter |
| puroR |
Puromycin resistance gene for mammalian selection |
| SIN/LTR |
3' self inactivating long terminal repeat |
| f1 ori |
f1 origin of replication |
| ampR |
Ampicillin resistance gene for bacterial selection |
| pUC ori |
pUC origin of replication |
| 5' LTR |
5' long terminal repeat |
| Psi |
RNA packaging signal |
| RRE |
Rev response element |
back to top
TRC II Vector - To be announced Soon.
MISSION Custom Cloning Vectors
pLKO.1-CMV-tGFP
There may be times when classic drug selection for stable or semi-stable cell lines is not possible or desired. There are the rare but documented cases of gene expression patterns changing due to drug selection even in the presence of a resistance marker.4 Cells may resist puromycin selection due to expression of endogenous resistance genes such as mdr1-pgp.5 In addition, drug selection methodologies require manual colony picking which can be both labor intensive and time consuming.
Sigma now offers pLKO.1-CMV-tGFP as a solution to these problems. This vector maintains all of the other elements of the pLKO.1 vector, but the puroR gene has been replaced by TurboGFP™. Sigma’s MISSION bioproduction team will customize the vector with either a TRC shRNA sequence or your favorite shRNA sequence. With TurboGFP in the vector, FACS and fluorescence microscopy techniques allow for rapid estimation of transduction efficiency and automated rapid selection of cell populations that stably express the shRNA.
pLKO.1-CMV-tGFP sequence (10 Kb txt file)
Vector Map
back to top
References
-
Stewart, S. A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 2003, 9, 493-501.
-
Zufferey R. et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol 1997, 15, 871-85.
-
Zufferey R. et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 1998, 72, 9873-80.
-
Su, L. H. et al. Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection. Mol. Biochem. Parasitol. 2007, 156, 124-135.
-
Pastan, I. et al. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4486-4490.
Send this page to a colleague.
Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: RNAi@sial.com.
MISSION is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Label License.
TurboGFP is a trademark of Evrogen Co.
back to top
RNAi Homepage
|
