Hi,
I am intending to do a CRISPR experiment where I knock in a GFP into a non-coding region of DNA into a non-dividing cell line using HDR.
The problem is, when i transduce my cells, both integrated and episomal cells express GFP. The cells do not divide so the episomal GFP expression cannot get diluted either.
The cells dont seem to be amenable to non-viral transfection, and i definately have to include a promoter in the HDR construct coz I am targeting a non-coding region.
How do I select out the cells which have integrated GFP as opposed to episomal? Or, I could destroy or turn off the expression of GFP in the episomal construct, but I dont know any way this can be achieved!
Many thanks in advance!
Hi Abel,
Nice and hard challenge!
First of all, did you check the ability of your cells to do HDR because non dividing cells are not the best type of cells for that and often the efficiency of HDR in this type of cells is very low. So even you have some HDR events, you have to obtains cells in which ALL the plasmides which are entred will be integrated maybe you can add some factors to enhence HDR.
To eliminate cells which contains episome you could design another expression cassette with thymidine kinase (TK) which has the advantge to allow a negative selection with a ganciclovir treatment The design will allow that after HDR, the expression of TK will be turn off.
In this way, if you do a gancilcovir treatment, all the cells which have episomes will died and the cells GFP+ which will survive should have only the integrated plasmide. But I think the issue is to have a system in which you can have 100% of integrated plasmids in one cells ...
Hope this will help you
Nicolas
Hi Nicholas,
No idea how to measure HDR efficiency either because like I said I cant distinguish the integrated from non integrating plasmid. Maybe I can do some genomic analysis.
To ensure all the plasmids are integrated I could use a lower MOI than 1. So the chance of multiple episome per cell is rare. Maybe I get lower number of integrated cells too, but then i just have to make more flasks!
But assuming I I do manage to get a small % HDR knocked in.
Great idea btw. Will definately try that. But I scared the HSV Tk also kill the small % of integrated as well coz gancyclovir can diffuse out of cell.
But you have any idea how to design the HDR construct that the GFP only turn on if integrate? Then its simpler!
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