I would like to generate stable knock-down of target gene expression in DLD-1 cell line by shRNA. I have pLKO.1 HIV- based lentiviral vectors, the selectable marker is puromycin.
Cells were transfected with Lipofectamine 2000. After 48 h I started selection by appropriate concentration of puromycin.
Two weeks later I checked expression of mRNA by RT-PCR. mRNA levels of the target gene did not decrease relative to the pLKO.1 empty vector. I have few questions:
1. Is it possible to cells express only puromycin resistance gene without target gene?
2. Is it possible to obtain stabile cell line without lentiviral transduction ?
If yes, how do it ?
3. Does anyone have experience with stable transfection of DLD-1 by pLKO.1 vector using lentiviral transduction ?
I'll be grateful for the help.
Hi Edyta,
I. Did you package the pLKo lentivirus, if not that could be the problem. You would not be able to silance your gene of interest by simply trasfect the pLKO in your cell line. You need to follow the Addgene protocol. I used HEK293T cells for packaging the lentiviral particles and cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and antibiotics. The infectious viral particles were produced as described in Addgene web site: http://www.addgene.org/tools/ protocols/plko/.
II. In order to get good silencing you need to use the following web sites:
http://rnaidesigner.invitrogen.com/rnaiexpress/design.do
http://www.thermoscientificbio.com/design-center/?redirect=true
I have used combination of these two web sites, but the final version was from the thermoscientificbio web site. Follow the advices from the Addgene web site: http://www.addgene.org/tools/ protocols/plko/ to build the hairpin and clone into pLKO vector.
Good luck,
Ivailo
to 1) yes by promoter silcengin it is possible that the shRNA is not transcribed any more but your cells are puromycon resistant.
2) in therory you can get by spontaneous homologious recombination a stable cell line but I have never heard about it for pLKO vector.
3)- no experience with this cell line, but as pLKO is a lentiviral vector there should not be any problem with the right helper plasmid...
Hi!
In theory, it should work, but in real practice, it doesn't.
I have to admit that I buyed a shRNA plasmid "for transfection" and results were not as the supplier promised me: low efficience, unreproducible results and low % of silencing.
You have to use lentiviral packaging in order to obtain a lentiviral particle to express your shRNA.
If I well remember, pLK0.1 is a 3rd generation lentiviral plasmid, so you need 3 helper plasmid and HEK293T for the correct packaging of virus. Is not too complicate as it can seem...
It is possible to stably transfect lentiviral transfer vectors without lentiviral packaging and transduction because pLKO.1 expresses the shRNA under the U6 promoter. http://www.addgene.org/10878/ But the main problem is obviously transfection efficiency. If you have ~30-40% transfection efficiency, you should have no problem getting your knockdown. Note that pLKO.1 is a large plasmid, so transfection efficiency could be low.
When I stably transfected a lentiviral vector into PC3 (prostate cancer) cells for miRNA expression (under a U6 promoter, the same as an shRNA), I obtained a few clones which were antibiotic resistant but not overexpressing the miRNA, a few with moderate expression, and a few with high expression. The expression of the entire pool was somewhere in the middle. So I'd give a try with individual clones of your puromycin-selected cells, not just the pool. (My guess is that stable integration is random for transfected plasmids, and the plasmid might get linearized at random locations, possibly destroying the U6 promoter and shRNA but leaving the puromycin resistance cassette intact. This may not be a problem with lentiviral transduction because genomic integration is directed by pol/Integrase and LTRs. But this is just speculation!)
Stable transfection of lentivirus or retrovirus vectors should NOT be a problem.... only thing which it may have a difference in expression of the gnene of interest in different clones.... so best will be to select single clones out of transfection and check each one of them, find the best one you think is the one....!!!!! I think you have to consider the difference between transfection efficiency and lentivirus transduction.... a difference of day and night on top of it viral particle can deliver the gene of interest here shRNA into the nucleus, whereas the simple plasmid transfection is very much hit and luck basis delivery into the nucleus.....!!!! so going through the problem you described, my guess is you haven't used enough of plasmid to transfect your cells I will sugget to use 10ug of shRNA plasmid to transfect a 10cm plate...... then next issue can turn out that you haven't gone high enough with Puromycin selection pressure....!!!!! keep increasing the selection pressure to max till you get the desired result...... hope these ideas help....!!!!! I have done both lentivirus and retroivirus transfections and generated many cell lines so I know IT WORKS......!!!!
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