Best regards! Has anyone has manipulated Caco-2 cells with CRISPR Cas9? Troubleshooting question :)

I am trying to make a Caco-2 Cas9 cell line by lentiviral delivery (pLentiBlast and Hygro) but I have been having issues with it.

I just wanted to ask if any has made this cell line or manipulated Caco-2s with CRISPRs, to know if it might be particularly resistant or maybe I just need to workout the protocol for it.

Many thanks in advance !!!

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Pearl Mummyover 7 years ago

I am just about to try to do CRISPR (but via transfection of pX458 plasmid) on Caco2 cells.
What issues did you have with them?? I kind of anticipate that clonal selection (growing them from the single cell) could pose a problem ...

Sienna Golemabout 7 years ago

Did either of you have success CRISPRing Caco2 cells? I'm interested in giving it a shot, any advice would be really appreciated.

Pearl Mummyabout 7 years ago

We are still growing the cells... After initial sorting and clonal selection we had some contamination of the cells, so we had to start it all again.
So far what I can suggest is after sorting to plate at least 25 000 cells in 10 cm dish to get some colonies. We also used Caco2-conditioned medium because we observed that it is difficult to get them growing when they are very diluted.
But then how efficient they are with Cas9 I cannot tell you still, we are waiting the sequencing results....

Cyan Sphinxabout 4 years ago

This is a 3 years post, but did you got your CaCo cells? Any suggestion for who is starting now? I will do my first try this week. I am planing to use electroporation and PEI methods, and 5ug/mL puromicyn to the selection.

Crimson Wyrmover 3 years ago

I was wondering if anyone got the CRISPR transfection to work with Caco2.

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