Question of puromycin selection in mouse cell line

[Jayci Huang]

Hi CRISPR experts,

I've got a problem, actually, in my recent experience of CRISPR-mediated gene editing. 

So I intended to do a 500bp knock-in in a mouse cell line N2a. I used pLentiCRISPR v1.0, which contains puromycin selection mark.

Then I did a kill curve as recommended, and decided a concentration of 4ug/ml puromycin, which is relatively high.

But afterwards I found the cell line was quite unstable for the selection. 

Sometimes a 4ug/ml puro can kill most/all of the untransfected cells (negative control) sometimes not. 

Sometimes it leaves a huge numbers of cells alive but I dont think the transfection efficiency is that high.

I have no idea why it happens. I've re-prepared puromycin and validate it in another cell line, it's working stably.

Has anyone who worked with mouse/rat cell line encountered similar situation? 

Do I have to increase the concentration or do I have any other ways to test where went wrong?

Any advice would be helpful.

Thanks!

Jayci

[stephen...@irbbarcelona.org]

Hi Jayci,

A couple of points

1) There is batch to batch variation of puro. Are you using the same batch? We have done a limited amount of work with N2A's and 1ug/ml seems to be sufficient to select, so 4ug/ml seems very high.

2) N2A's transfect very efficiently (with lipids, nucleofection etc.) so it may not be suprising that you have a large number of cells surviving

Best,

Steve