Hi there, I am attempting to transduce cells with the TLCV2 plasmid containing our sgRNA of interest. My transfection of the HEK 293T cells seems to yield positive results as the EGFP is clearly expressed by my cells under the microscope. I harvest the media at 48 hours and then allow my target cells to sit for 24 hours without disturbing them. I then selected them with puromycin for 3 days at 4ug/ml which is well above what is needed to kill the controls but I have been concerned about residual non transduced cells. After selection though, when I go to image my cells they do not express the EGFP gene. This has been shown to be previously successful by members in our lab so I am at a loss for the cause. I take the media directly from the 293T cells, spin it, and filter with .44 filter. I do not concentrate the virus at all. I have yet to run a western blot to see if my GOI is knockdown as I do not want to waste a large chunk of time if I can know my transduction did not work from the EGFP.
Does anyone have experience with this issue? If so how did you resolve it? Any help is appreciated thank you.
A quick glance at the TLCV2 vector from addgene...it's an all-in-one tet-inducible....have you tried adding some doxy to your transduced cells? According to the map, the backbone expresses Cas9 2A GFP...so your GFP will be under doxy induction as well.
As these promoters are sometimes leaky, you are likely seeing GFP in your packaging cells because of the quantity of DNA you transfected (many per cell). Viral transductions are much less, and therefore, you may not be able to visualize the small leakiness.
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