Puromycin selection for pSPCas9(BB)-2A-Puro v2.0

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Neuropath

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Sep 27, 2016, 11:38:35 AM9/27/16
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Hi CRISPRians,

I am using pSPCas9(BB)-2A-Puro v2.0 to knockout a gene in human ES cells. Can anyone share their experience as to how long to treat the transfected cells with puromycin to select for transient transfectants but not stable integrants? How long after transfection should I start puromycin treatment?

Thank you.

~neuropath~


Mark

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Sep 28, 2016, 10:52:22 AM9/28/16
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From personal experience I would recommend that you keep the puromycin on no longer than 2 days, and I usually change to puromycin media about 1-2 days after transfection.

Might be different in your cell line, but that's a starting point that's worked well for me.

Mark

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Sep 28, 2016, 10:53:33 AM9/28/16
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Just to add, I usually have a control mock transfected well included with each experiment so I can monitor cell death relative to the puro-R transfected cells.

Neuropath

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Sep 29, 2016, 12:37:14 AM9/29/16
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Hi Mark,

Many thanks for your suggestions. I will follow them.

Regards,
~neuropath~

Andy Cho

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Sep 29, 2016, 5:27:25 AM9/29/16
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Hi Mark,

May I ask you what concentration of puromycin do you use in selection?
I used 0.5 ug/ml puromycin select for 24 hours, but the iPSC all death after electroporation.

Thank you.

Nadine Schrode

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Sep 29, 2016, 10:14:32 AM9/29/16
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How long after electroporation are you adding the puro?

Andy Cho

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Sep 29, 2016, 9:49:11 PM9/29/16
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Just one day after electroporation, I add 0.5 ug/ml puromycin into the medium for another 24 hours.

Mark

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Oct 3, 2016, 11:34:38 AM10/3/16
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Hi Andy,

Sorry for the delay, I need to fix my notifications.

I used 2 ug/mL puromycin for NIH-3T3 cells so the concentration will be cell-line dependent. Looks like iPSCs are quite sensitive, or perhaps the delivery is poor and there is no resistance if they're all dying. I would suggest trying adding the puromycin perhaps 48 hours after electroporation rather than 24 hours...might be too early. These guys (http://www.nature.com/mt/journal/v23/n3/full/mt2014226a.html) seemed to add puro 96 hrs after nucleofection, but they are looking for stable integration it seems... so perhaps 48 hrs is a good time point to try. They used the same concentration as you did.

I hope that helps,
Mark

On Thursday, September 29, 2016 at 5:27:25 AM UTC-4, Andy Cho wrote:
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