Puromycin selection kills all cells in endothelial cell line transfected with px459 ?!!

Rex

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Apr 5, 2016, 5:00:15 PM4/5/16

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Hi everyone,

I am trying to screen clones using crispr in endothelial cells (EA.hy926). I transfected two px459 based plasmid in order to delete an exon from a gene. I added puromycin contained culture medium 48 hours post transfection. BUT, puromycin (even as low as 25ng/ul) kills all cells!!! Is there someone who ever used this cell line? Can you please let me know your method for screening clones using EA.hy926 cells? I've failed twice, very frustrating... Thanks!

Rex

Multi Nucleated

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Apr 5, 2016, 5:47:57 PM4/5/16

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25 ng/ul as in 25 ug/ml? That's a bit high from what I've seen used (0.5-5 ug/ml range)...

YIng Dang

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Apr 5, 2016, 6:12:29 PM4/5/16

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Make sure you use pX459v2 (62988), not pX459 (48139)

Rex

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Apr 6, 2016, 9:46:11 AM4/6/16

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nanogram... it's extremely low for most cells...

Rex

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Apr 6, 2016, 9:48:05 AM4/6/16

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Is there something different with these two? I am a new guy in this field.

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Rex

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Apr 6, 2016, 9:57:12 AM4/6/16

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I just checked, we are using 62988. I don't know if this is due to the sensitivity of endothelial cell??!

On Tuesday, April 5, 2016 at 6:12:29 PM UTC-4, YIng Dang wrote:

YIng Dang

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Apr 6, 2016, 12:20:10 PM4/6/16

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If you are sure about your Puro concentration( let's say you have killing curve data), then you probably should also double check your transfection efficiency.

Rex

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Apr 6, 2016, 2:57:43 PM4/6/16

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That's good point! I'll check it and let you know once I have the result. THANK; )

jiangbin wu

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Apr 8, 2016, 1:27:05 PM4/8/16

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Hi, Rex, Haven't seen you for a long while. Actually, we are using three kind of way to do gene known down with CRISPR/Cas9 technology. One is just like you, transfection then use puromycin to get the stable cell line. The other is using spCas9 stable cell line for gRNA library screen. I think the third one may be suitable for your situation. Normally we don't use puromycin for stable cell line screen in this way. We transiently transfected cells with Cas9/gRNA expression vector and culture cells for 2-3 days to let knock-out happen. Then we trypsinized and gradient diluted cells to get the mono-colony. Usually we use two gRNA at the same time so that we can simply use PCR to find the deletion colonies. Of course low efficiency is a problem for this way, maybe lower than 10%, so you need to check more colonies compared with stable cell line. But this way avoid some off-target effect we don't know in stable cell and that's why we are still using this way. I think you can try this way in your system and take the transfection effiency in your endothelial cells. Of course you still can try to use puromycin in a lower concentration based on your killing curve data.

Rex

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Apr 8, 2016, 3:37:13 PM4/8/16

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Wow!!! How lucky to meet you here!!! Your strategy looks great! I will try it and let you know the outcome ; )

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