Losing GFP signal after cell sorting...Have this happened to anybody else?

[Jume J]

Hi everyone,

I have successfully transfected my cells with pSpCas9(BB)-2A-GFP (PX458) carrying my gRNA. After transfection, I sorted for GFP+ cells by flowcytometry. However, after sorting the cells seem to lose their "green" color. I'm pretty sure that the sorting has been done properly and we selected only the cells that showed distinct GFP intensity. Have this ever happened to anybody? Is it possible that even though the GFP is not present, Cas9 might still work. I'm concerned because they seem to share the same promoter. Please let me know what you guys think? and what would be your suggestions?  

Thank you!

Kornkamon (Jume)

[Anoeska]

On what time are you losing signal?

And is the aim of your experiment to transfect cells stably or transient?

In transient expression I would expect to loose GFP signal because you loose the plasmid after a while. This shouldn't be a problem for Cas9 activity: transient transfections are 'long' enough to get nice CRISPR activity. 

Anoeska

Op zondag 6 juli 2014 23:06:36 UTC+2 schreef Jume J:

[Jenny Shin]

Actually I was wondering about similar sort of things as well.

Can I ask when you start to see the expression of GFP and when it is gone (how many hours/days after transfection)?

Also which cell did you use?

I am planning to transfect hESCs with puromycin resistant plasmid (px459) and wondering when I should start to treat with puromycin and when to stop since the resistance will be transient.

Jenny

[Jume J]

Dear Anoeska, 

Thank you for your reply. I tried to make a population (by sorting to enrich the GFP+, can it be stable?) of Cas9+ cells, so I transfected my cells with GFP-CRISPR plasmid. Then, after 48 hrs of transfection, I checked for GFP+ cells and saw that there are some in my transfected plates. After I sort them, 24 hrs later, the cells attached well but I didn't see any GFP signal. However, I started seeing "some" GFP+ cells in the sorted populations 72 hrs after sorting (5 days after transfection). Then, I wait for them to get confluent, so I can use them. However, the signal disappear again after day 7 post transfection. So, I'm wondering whether the CRIPSR is still there or not? Does it mean my exp have already failed? or it might still be there and working even it shares the same promoter with the GFP? The objective of my exp is to have a population of cells that have my gene of interest mutated or knocked out. Now, I still have them growing, but also start the next set of exp with the Puro-CRISPR, so I can select them by puro. 

Thank you, 

Jume 

[Jume J]

Dear Jenny, 

you can read my reply previously for the timeline detail. I transfect the CRISPR to bone marrow stromal cell (HS-5). Personally, I start seeing a lot of my GFP signal at 48 hrs after transfection. 

Jume

[Anoeska]

So what you're seeing could be:

48-72 hrs post transfection: expression of the plasmid is sufficient to detect the GFP (and CRISPR should be working). Depending on the transfection and/or plasmid, I usually wait 24-48 hr to detect anything so this would be normal I guess.

7 days post transfection: you start to loose GFP because the cells are loosing the plasmid (transient transfection). This means from this point on there is no CRISPR activity anymore but you should already have cells that are mutated. You don't need constant expression of CRISPR, the mutations made between 2 days - 7 days post transfections are permanent. You then have to create single clones from your cells and check these for desired mutations to select a population with the wanted mutation.

Use of the Puro-plasmid could help you to get a higher percentage successful mutations: you kill the cells that are not transfected (this doesn't mean all those cells have a successful mutation). Make sure to quit Puro treatment before the cells start losing the plasmid (which in your case would be around 7 days).

Hope this helps :) 

Op zondag 6 juli 2014 23:06:36 UTC+2 schreef Jume J:

Hi everyone,

[Jume J]

Dear Anoeska, 

This definitely help! Thank you so much!

Jume