Stable Mammalian Expression with Retrovirus - (Feb/24/2010 )
I am transducing mammalian hepatocellular carcinoma cells with a IRES-GFP MMLV-based retrovirus construct containing a 1.5 kb insert.
I've read (in non-peer-reviewed publications) that stable expression/insertion into the target genome (i.e., the mammalian chromosomal DNA) can occur in as little as 3 days. First, does anyone know if this is generally true or not?
Second, I want to FACS sort the GFP+ cells as soon as possible after infection/transduction, but I want to also make sure that the ones that are sorted are stably expressing the DNA. Does anyone have any suggestions on how many days/weeks to wait after transduction before FACS sorting my transduced mammalian cells with the goal of having sorted stably expressing cells?
It is possible to make stables using a retrovirus plasmid or even a virus. You will have to select them for a long time anyways.
As long as they express GFP, you could sort them but its better to select them before running them through FACS
scolix on Feb 24 2010, 02:14 PM said:
As long as they express GFP, you could sort them but its better to select them before running them through FACS
Why not transfect a GFP plasmid in without selection and see how long the fluorescence lasts? You will probably need to go out 7-10 days to be sure your GFP is stably integrated.