Hi, I was wondering if anyone could recommend me or perhaps suggest to be a good plasmid for overexpression of a gene in human cells?
I want to create a stable line of neuroblastoma cells (SH-SY5Y and/or IMR-32) and endothelial cells (hBMEC, TY10, or CaCO-2) which overexpress my human gene of interest and i'd like to use lentiviral transduction to get the best efficiency. I also would like the gene untagged as I'd like to observe normal function when upregulated.
From a literature review i've decided that the most important thing is going to be choosing the promoter. I would prefer to select by puro/neo/blast etc rather than using a cell sorter and fluorescent marker protein, but either would be OK, I would also prefer it if the cells didn't express any fluorescent protein at all because i'd rather they don't transcribe anything unnecessary - but i'm not overly fussed. The most important thing is the right promoter.
So i've gathered that CMV is a general 'one-size-fits-all' and works for most things, but that some promoters may work better for specific cell lines. The U6 promoter is reported to work really well with neuroblastoma cells, while CMV is more modest in action. CMV seems a pretty good fit for endothelial cell lines, though I did find a paper that found U6 was really good in the endothelial cells of mice. One thing i'm particularly interested in is possibly using tet-on overexpression, has anyone found this to be effective in the cell lines i've described? If tet-on is an effective promoter then that would definitely be the best choice for me.
Can anyone recommend me a plasmid they have used which is effective? So far i've narrowed it down to pLJM1-EGFP (CMV), and pCW57.1 (tet-on). I've not been able to find a U6 promoter that isn't for shRNA that isn't from a commercial company and extremely expensive.
Most importantly .. am I going the right way if I want to run experiments to see the effect of upregulation of my gene on cell function?
Thanks for reading.
:EDIT: I should add - the backbone should be 2nd or preferably 3rd generation because of safety concerns. Otherwise I would use pBABE as it seems widely used, though appears to not be 2nd/3rd gen and produces viral particles without needing to be transfected with packaging plasmids.
Edited by TheFOrsh, 28 February 2015 - 07:45 AM.