I'm a 1st year graduate student. I've just constructed a new retrovirus vector which holds CreERT2 system. Now I'm considering about checking it's function by transfecting it to ES cells which has certain floxed gene, and knocking the gene out conditionally with tamoxifen treatment. I searched the way to do it, but most of the reports are based on in vivo knock out. Could anyone recommend a protocol of doing that in vitro using ES cells? Thank you!
Dear Daisuke Koga,
did you already transfect your ES cells with your vector?
If so, you have to check, if your construct is integrated into the genome by sequencing.
Then, induce your cells with tamoxifen for at least 48h (up to 96h), afterwards you harvest your cells for DNA and RNA isolation.
Simultaneously, you also harvest untreated cells as a control.
Then, you have to check, if your floxed gene is really cut out of the genome by sequencing and don't forget to compare it with your control.
To further confirm your KO, you have to perform qPCR and Western Blotting.
I hope this helps.
Best, Lisa
Dear Lisa,
Thank you so much for your advice.
Actually, I haven't transfected ES cells with the vector yet.
My one of my lab member has just tried it, and it seems to be difficult, although it was successful using NIH3T3 cells.
What do you think about using PCR instead of sequencing which you mentioned?
Best regards,
Daisuke
Hi Daisuke,
here's a paper in which they are doing something close to what you are mentioning. The protocol for tamoxifene treatment of floxed mESCs carrying a stable CRE-ERt2 construct is described.Just check the m&m section for concentrations and exposure time.
Article Beta-Catenin Is Vital for the Integrity of Mouse Embryonic Stem Cells
Hope it helps
Francesco
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