Hello everyone! I will summarize my situation: I am performing KO using CRISPR/Cas9 for PTEN in Prostate Cancer cells (DU145 and 22RV1). I am using a commercial vector that includes the guides, Cas9, puromycin resistance and GFP. I have infected the cells with the virions carrying the guides, I have done the selection with puromycin and I have even done a first western blot of PTEN. In this Western of the cell pool I have seen a large decrease of PTEN with respect to the Wildtype cells, which means that it has worked to some extent and now I must isolate clones to be able to obtain a complete KO. However, when I have gone to look at GFP expression on the fluorescence microscope I have not seen any of the green cells (I have also used GFP expressing cells as a positive control to make sure there is not a glitch with the microscope). I don't quite understand why you see a decrease of PTEN in the WesternBlot but I am not able to see GFP if in theory they go in the same vector (The vector is Merck's LVL01). Can anyone know what is going on? is there any explanation? should I start again from the beginning? Thank you very much in advance
Dear Miguel Martín Serrano ,
I'm not familiar with your exact system. But it might be the case, that the GFP is just your positive control for the transfection/transduction. Later when the virus is diluted by proliferation and selection the GFP signal is gone, since its only expressed while the vectors DNA is still present in the cells.
I would assume that when PTEN is gone, that the KO was successful.
You might want to try to amplify the site of your frame shift via PCR from genomic DNA, clone it and sequence some of the clones.
Best wishes
Soenke
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