Hi,
I made a construct using mouse specific shRNA driven by U6 promoter, it did not show the knockdown of my target gene in NIH/3t3 cells.
Same shRNA sequence used by another group, works in mouse cells (paper published)
Only difference is the promoter, the study has used human H1 promoter (Ambion pSILENCER H1 shRNA vector) and I have used same shRNA with U6 promoter.
Does anyone have any thoughts about differences in promoter particularly for mouse cells. Please share your suggestions.
Apoorva
Does your shRNA construct start with G or A? These nucleotides are required for efficient transcription from U6 promoter. Otherwise, both U6 and H1 promoters should work in mouse cells with U6 being a bit stronger promoter.
Article Mutation of nucleotides around the +1 position of type 3 Pol...
Article Stable RNA interference: comparison of U6 and H1 promoters i...
Dear Evgueni,
Many Thanks for your suggestions. My shRNA seq starts with a G.
The articles you sent are very helpful and appears from the study that U6 is a strong promoter for siRNA than H1.
I don't know why its not working in my case
Regards
Apoorva
Check your transfection efficiency, maybe there is a way to enrich for transfected cells. Assuming that there are no technical errors, the half-life of your protein of interest might be quite long, and you have to wait longer to observe a detectable depletion. Are you seeing any reduction on RNA level? Also, make sure that your cells, compared to cells in a published study, do not carry a SNP at the shRNA target region.
hi, Dr. Apoorva Verma
both the U6 and H1 promoter work fine for shRNAs, though do not work in glioma cells. One thing to consider is that if you want an inducible promoter, and H1-based promoter is probably better
i suggest you more read these:-
1- Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain
Article Stable RNA interference: comparison of U6 and H1 promoters i...
2- Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain.
Article Stable RNA interference: comparison of U6 and H1 promoters i...
3- Development of a tightly regulated U6 promoter for shRNA expression
Article Development of a tightly regulated U6 promoter for shRNA expression
4- Pol III Promoters to Express Small RNAs: Delineation of Transcription Initiation
Article Pol III Promoters to Express Small RNAs: Delineation of Tran...
5- Regulation of U6 Promoter Activity by Transcriptional Interference in Viral Vector-Based RNAi
Article Regulation of U6 Promoter Activity by Transcriptional Interf...
good luck
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