I am trying to make stable cell line (HEK293T) using CRISPR/Cas9 technology. I co-trasnfected CRISPR plasmid along with donor plasmid (carrying Tet-on inducible GOI and GFP + constitutively expressed BSD resistance gene) and did BSD (blasticidin) selection for more than 2-weeks (16 days). The treated cells survived unlike control cells (non-treated). I induced cells with 2ug/ml DOX but no GFP signal. Considering degradation of my GOI and GFP, I did PCR using genomic DNA of positively selected cells (as template) but no amplification. I also run PCR for positive control (donor plasmid) which showed amplification. I am wondering (1) if there is no integration of my sequence of interest then why there is positive drug selection of my cells? (2) Has my sequence been integrated into the genome but I don't have skills to find out? Can someone guide me please!!
I suggest to do Sanger sequencing. If the result is in agreement with PCR, there is no integration of your template for sure. It’s highly possible that there is no integration. 1) I feel the problem is the blasticidin selection. After the selection, the control should be all dead while the CRISPR treated should some survival cells. Sometimes, the false positive may happen. 2) Do you run GFP plasmid negative control to make sure the electroporation or lipofections works?
look at this
https://www.researchgate.net/post/Why_is_my_G418_selection_not_working
cheers
I could be that the cells just take the Blasticidin resistance gene...
or it could be that your PCR is not working on cell extract (more inhibitor than in just plasmid pure DNA) Did you try Phenol Chloroform extraction on your cell lysate? It could help a lot
good luck
Dear Lin,
I applied optimized PCR conditions if it fails then don't you think Sanger sequencing is worth doing. (1) Yes all control cells were dead (2) Yes I confirmed success of lipofaction by using control GFP plasmid.
Dear Pejman,
Thanks for link.
Dear Marie,
I used SP-DNA extraction kit for non-treated and treated cells and it gave me positive PCR without insert. It means template quality is good for PCR.
Do you try to subclone your surviving cells? Anyway, the selection is a bit short mightbe.
Thanks to everyone for your kind suggestions. I got the positive PCR after selection. Now, em confirming by Southern blotting.
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