A donor DNA for homologous recombination after a Cas9 cleavage must have an antibiotic resistance gene?

25 August 2022 1 5K Report

I'm trying to establish a bovine knock-in cell line using CRISPR-Cas9. I designed a donor DNA containing the transgene, a GFP gene and the homology arms. Should the donor DNA have a antibiotic resistance gene? The molecular vector to cas9 and gRNA delivery have an ampicillin resistance gene and a GFP gene.

Robert Adolf Brinzer

If you have a cell sorter you could sort based on GFP. Still having a mammalian selection marker eg. for G418, puromycin or blasticidin resistance would make selection easier. You can even have the marker flanked by recombination sites for recombinase mediated removal of the marker after stable integration.

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