Andrei,

Thanks much for this feedback. I was not aware that the shRNA plasmids are expected to have problems with transient expression, as you describe. Is there a reference or explanation so that I can educate myself as to the issues here between lipid based transfections and electroporation?

Thanks again. Nothing is better than first hand experience. We don't have ready access to electroporation so will make a few viruses and if they work, after the lipofectamine transients did not, then we will know for sure.

Transfection efficiency is very critical for any shRNA-based knockdown. We usually make sure to get more than 80% of cells transfected before we check the knockdown of our proteins. Viral delivery would be a more reliable way for shRNA knockdown. However, pLKO-TetON is somehow leaky. You may get trouble if you plan to establish a stable cell line to knock down an essential gene. In our hand, we could see a very nice knockdown for one protein shortly after we infected cells. However, all cells survived from 2-week of antibiotic selection lost the knockdown.

First, transferring sequences that work as siRNA into a SHRNA delivery vector is not always optimal. The processing of the shRNA into siRNA in the cell by Dicer is not perfect and the desired siRNA may not be the most abundant sequence generated.

The pLKO-TetON vector has not worked in our hands all the time when transferring sequences as you are trying to do (probably about 80% of the time so far), however we are going through the virus integration route.

There is some information we have publically available at http://www.broadinstitute.org/rnai/public/

I suspect the transcription start site in pLKO-TetON is not as expected. The same 19-nt (targeting sequences) shRNAs in a regular non-inducible vector worked very well, but didn't work in pLKO-TetON. After we extended them to 21 nts, they knocked down pretty well with doxcycline.

Nice site with plenty of useful information. Thanks, Glenn. Still working on this and still not completely clear to me why transients transfections don't work to test the pLKO plasmids before going into lentiviruses. We have used pSUPER for transient siRNAs with good success in the past.

to overcome the transfection issues (some cells are hard to transfect), you may opt for a selectable shRNA expression vectors. i.e. pSilencer™ 2.1-U6 hygro or puro has been very helpful for our studies since we know both Lipo and Transfection method have not getten even 20-25% for our cells. The obvious drawback is, it takes some time to select cells but the positive side is you always get consistent results once selection is done.

Lentivectors are not used for transient transfections, they are used to produce virus particles by transfecting a producer cell line (such as HEK-293T). These virus particles can then be used to infect cells. If you wish to have an inducible system, you may need to use two different lentivector systems.

The Clontech website has a variety of tools and different explanation about how this works, and you may want to check this out.

http://www.clontech.com/US/Products/Inducible_Systems/Tetracycline-Inducible_Expression/shRNA?sitex=10020:22372:US&PEBCL1=KiPRVCPu0YF7ggw69uKvgLuMxi&PEBCL1_pses=ZG55F3842B2F42BF3FF4CF7A20A9E656E62F8192D7D0E6665768504B5E9CDF9A407F2B332CF5DBEE82A9D9F249D7F4DAB17C0EA79534EC73AC

I hope that this helps.

I have a question about this topic

if i have specific amino acid sequence in a peptide chain and i want to suppress the expression of the 11 amino acid in the peptide chain how can i detect the miRNA or siRNA which will make this activity

There are a lot of resources available on the Addgene site (including all kinds of vectors for siRNA studies). here is one example http://blog.addgene.org/your-lentiviral-plasmid-faqs-answered but the home site addgene.org has lots of info too.