I am trying to knockin a DNA fragment (having Blasticidin selction marker) into the genome of nHDF (normal Human Dermal Fibroblast) and MEF (Mouse Embryonic Fibroblast) cell lines by using CRISPR/Cas9 technology. I want to select positive clones using blasticidin selection.
For how long time, the transfected plasmid transiently survives in relevant cell line? When should I apply blasticidin antibiotic selection to minimize the false-positive selection due to the expression of blasticidin resistance gene from non-integrated DNA (plasmid)? Thank you!
Hey,
I aim for a 48 hour antibiotic selection, but you should really just make your own resistance curve. Cells with transient plasmid would keep the resistance for a couple of days. After which you should see a drop in the number of live cells in your culture. Note that not all cells will die, since persistent selection will lead to random uptake of the plasmid into the cell DNA
Thank you Yeshai! I have started experiment to make my own resistance curve according to my requirements.
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