I’m using a miR30 based shRNA lentiviral system, namely the vectors pGIPZ, pCMV-dR8.91 and pCMV-VSV-G, in order to knockdown BV2 and N2a mouse cell lines. My current results are not encouraging since I saw no decrease of the gene expression using Real-time PCR and more important no GFP is detectable with fluorescent microscope after 4 weeks of culture with the antibiotic of choice, puromycin. However the cells maintain their resistance against puromycin. So my questions are:
Does anyone know if all of these vectors are compatible with mouse cell lines and has anyone used them successfully before to knockdown mouse cells?
In a precedent post it was mentioned that GFP expression is diminished when the shRNA is being effectively processed due to their existence in the same transcript (http://www.protocol-...n-mirna-vector/). Wouldn’t that cause loss of resistance too? Admittedly I lowered the antibiotic concentration to help in raising individual cell clones. Could this have caused loss of viral vectors gene expression?