How do you use transfection method? If you use virus, you may be success to this experiment. If you use lipofection, you may be fail to this experimet, because some shRNAs are not stable on the lipofection.

I work in primary human melanocytes and tried to used lentiviral vectors to silence a transcription factor of interest. We also tested out the polybrene concentration as well as the viral load for transfection of our cells. Then we tested out different puromycin concentrations to make sure that a significant number of cells would survive for propagation. Unfortunately, we tested using vectors that expressed GFP. Once we used the actual vectors for the TF silencing, the cells didn't respond the same way. Human melanocytes are very difficult to transfect and because they are established from different donors, each cell strain responds differently. At first, the cells took up the vector because they grew in the presence of puromycin, but when we tried to verify silencing by WB, there were no differences in expression. Additionally, some of the melanocytes became very, very sensitive to the same concentration of polybrene that we had been using previously. The cells would receive the polybrene/virus mixture in media and the next morning, everything was dead. How many cells are you trying to transfect? What kind of MOI are you using for your transfection?

Hi Anne,

I work with neural stem cells and I have some experience using shRNAs to downregulate the expression of my gene of interest. The resistance for puromycin is driven by a different promoter than your shRNAs. I would say that you may have a problem with the expression of your shRNAs. Check the promoter of your shRNAs to see if it can be the problem. I use a tetracycline-inducible promoter (pTRIPZ lenti-vector) which gives me good expression of my shRNAs. But there are many good promoters that you can use with you shRNAs. Some promoters work better that others depending on your cell type.

You could also try to clon a shRNA for GFP in your lenti-vector and see if it downregulates GFP. This is one of your best controls to see that your system is working.

Hope this help your research!

Best,

Ale

Thank you very much for all your comments.

Dear Takeshi Ueha: I used lentiviral particles to transfect in my cell lines by using polybrene.

Dear Anne, Alejandro: I used pRSI12-1 vector that containing U6 promoter and RFP (red fluorescent protein) gene. Actually, I didn't calculate the MOI as well as TU. I only constructed lentiviral particles in 5cm plate scale, then harvested virus particles, divided to 2 parts, each one used for transfecting to 10^5 cells. After finishing Puromycin selection, I selected the alive cells for my next experiment. I also used this vector to produce another shRNA that targeted another gene, and it worked well.

My problem is that I couldn't explain why only in 1 shRNA, most of the cells gradually became dead right after puromycin selection (no change in RNA expression). I checked transfected efficiency by immunofluorescent using RFP Ab, and it was high.

Do you have any idea?

Sincerely,

Uyen