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?
0
2 replies to this topic
#2
-
- Moderator
-
- 326 posts
a student
Posted 24 February 2010 - 11:14 AM
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
As long as they express GFP, you could sort them but its better to select them before running them through FACS
#3
-
- Active Members
-
- 223 posts
Veteran
Posted 24 February 2010 - 11:49 AM
scolix, on Feb 24 2010, 02:14 PM, said:
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
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.
Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley
