Hello.
I have a CRISPR knockout experiment designed for FAC-sorting my transfected cells (GeneArt OFP kit). Due to some events I may not be able to use the FACS anymore. Now I'm trying to use Homology-directed recombination method to introduce a selectable marker (puromycin) for my modified cells.
Is there a good plasmid to introduce the left and right homologous arms flanking a puromycin resistance gene?
Thank you.
Hello Denis,
A bit late maybe for your question.. But I have been looking into this recently as well. I think there is a nice option described in the following paper:
https://www.ncbi.nlm.nih.gov/pubmed/?term=18546598
It has been used for example in this paper:
https://www.ncbi.nlm.nih.gov/pubmed/?term=26826926
I think this would be a good approach. You can also purchase only the vector for the homology arms:
https://www.systembio.com/genome-engineering-precisionx-HR-vectors/overview
It might be nice to use the approach described in the Stem Cell Research paper in which also the tk is included in the donor plasmid.
Ann
Thank you for your answer, Ann.
I'll most certainly consider this method. Although I'm more inclined now to deliver the HDR template outside a plasmid. I'll probably try both ways.
Denis
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