Hi all. I came across a technical question from one of my friends, that whether a given culture of hESCs can be inserted with three different reporter constructs using CRISPR/Cas9 method, which means a triple knock-in line hESC. I could not find any relevant answer from the literature that I came across and thought researchgate would be an appropriate platform. Hence, I request all to give your inputs in this regard. Thank you.
Hello Wulligundam.
In theory, possible to KI in order of first target gene with GFP/FACS sorting enrichment, second target gene with RFP/FACS sorting by using the first clonal cells, and the third target gene with CFP/FACS sorting enrichment by subsequent using of the first and second target genes clonal cells.
However, undifferentiated hES cell line with the three KI should be established, all target genes should be house-keeping genes or undifferentiated hES cell-specific with the suggested procedure.
After the first or second gene KI, it would be possible to use other cell lineage-specific (i.e., neuron-, cardiomyocyte, beta-cell-) gene KI construct DNA transfections followed by one drug-selection or another drug-selection to isolate cell lineage-specific phenotypes after differentiation of hES cells.
Regards.
Hello Sang Ho Lee.
Thank you for your reply. The method suggested by you is feasible. But, my concern is that will hESCs remain pluripotent by the end of third knockin? Does the multiple rounds of transfection/microporation, sorting, selection, etc. which are all kinds of stress in one or other way, have any negative effect on their pluripotency and stability?
Hi.
You did not mention any concern!
However, you are not deleting any genes, but inserting genes (reporters) at the end of 3'-UTR of specific genes instead of randomly inserting genes in traditional plasmid DNA transfections in which many copies of tandom repeats are usually inserted. So the stability will be gradually settled down or lose after cell generations.
No worry about the pluripotency or stability of the KI-targeted a single copy of reporter. The only worry would be weak fluorescence due to the a single copy of gene insertion unlike traditional transfection. Especially, when the house-keeping genes are making a few copies, i.e., non-abundant protein genes.
Since no one is interested in triple KI of ubiquitous gene reporter-hESC line, no report.
Cheers.
Dear Sang Ho Lee,
It is a detailed clarification. Thank you very much. :-)
Regards.
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