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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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shRNA problems
Posted by: msp27 (IP Hidden, New member, 1)
Date: October 21, 2005 04:52AM
Hi guys,
I am trying to downregulate a B cell-specific gene using an shRNA driven by an H1 promoter. For this, I am using a retroviral vector that also expresses destabilised GFP and PuroR from a single bi-cistronic (IRES) mRNA driven by a CMV promoter. I first tried sorting GFP+ cells 3 days after infection and checking the levels of the gene of interest using RT-PCR. This way, I found a 7.5-fold reduction of expression. However, for the purpose of my experiment I need a lot of cells, so I tried producing a stable line. I repeated the infection using the same stock of virus, but this time I used puromycin to select for infected cells. In approx. 10 days, I checked the GFP expression and the absolute majority of the cells were GFP-positive. However, RT-PCR showed that my gene of interest was no longer downregulated at all! The levels of expression were exactly the same as in the wild type. It looks like the H1-driven shRNA 'gene' got downregulated or recombined over time. But is it a usual thing to happen with H1-driven constructs? Or maybe you have any other ideas as to why this is happening? And most importantly, can anything be done about it or I'd rather just look for a different vector? If so, which one would you recommend? Thanks a lot!
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