Can anyone recommend plasmids to be used to generate lentiviruses to be used to make stable cell lines? I have a few genes of interest I would like to clone into the expression vector and would like a recommendation of which plasmids to use. Thanks!
We are using cell lines for these experiments. But if you have advice for primary cells I would appreciate that as well.
I LOVE the pLEX vectors from David Root's lab
https://www.addgene.org/41392/
I create entry clones using Eric Campeau's entry vectors http://www.addgene.org/Eric_Campeau/
http://ericcampeau.com/
and recombine into whichever pLEX I need. I've been very happy with pLEX307.
https://www.addgene.org/lentiviral/ Check out this special collection of lentiviral plasmid from a non profit repository. This site also contains good protocols and textbook materials. If you prefer outsourcing I can recommend http://www.cyagen.com/service/lentivirus-packaging.html to you . They are quite cheap and you can order custom made plasmids there.I was satisfied with their service.
Regards,
Tomasz
Hi Kyle Potts,
I always used Plk0.1 vector to produced lentiviral plasmids. This is easy and use as a selection marker puromycin for your good knockdown or over expression of your specific targeted genes.
Here is 2 attachments to easily catch up protocols..
Best Wishes
Panneerselvam
David Root is on the TRC. The pLex and pLix are based off pLk0.1 (which his lab also designed)
Isn't pLK0.1 Designed for shRNA?
The beauty of the pLEX vectors is they ave options with EF1a and PGK promotors available. Cf CMV.
I am using pCDH-CMV-MCS-EF1 Hygro cDNA cloning and expression vector from SBI system Biosciences, it is very good. the company has different vectors they could be selected by different marker also some vectors contain P2A (or T2A) peptide and two MCS so you could express two genes under one promoter, such as our lab used it one our target gene and another one is CFP gene, so you could create stable cell line using FACS sorting (CFP ) after infection
The recommended vectors suggested above I agree with. I have stably transduced mouse ear fibroblasts and HUVECs and passaged 5X post puromycin selection. Passages are cryo-preservable
Do you want to overexpress or to knockdown genes that you interest ?
for overexpressing genes, i sugguest pSin-EF2-puro vector which gene can be driven by polymerase II
for knockdown genes, i sugguest pLKO.1-puro vector which composed of U6 promoter.
these two kinds of plasmids can be found on https://www.addgene.org
I agree with Yunghong, that pCDH (from SBI) works great. I use pCDH-CMV-MCS-EF1-GFP-T2A-Puro. This lenti gives me higher transduction rates than pLEX (in keratinocyte cell lines). I use second generation packaging - pMD.G & psPAX2. I can send you a protocol if you decide to go this route. Good luck!
We have used the pINDUCER system described by Meerby et al, (2011) PNAS 108, 3665-3670 to make shRNA knockdowns of a number of genes. It works very well and the knockdown is induced by doxycycline so you can have a cell line with normal expression and then use dox to reduce or eliminate expression in the same cells.
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