i'm doing Knockout of gene TRIM58 in CD34+ cells which are then going through Erythropoiesis to see if the KO has an impact on the enucleation process.
So i start with Hematopoietic stemm cells have them 3 days in cell expansion medium (SFEM2) and then i do the Knockout with CRISPR 15qg Cas9/1x10E6 + 8qg sgRNA/1x10E6 to make a cut in Exon 3(out of 5 exons that are in TRIM58 sequence) and 10 minutes after nucleofection(LONZA 4D, EO 100 program) i add 2 Adenoviruses (MOI=2x2500). One AAV has Left and right homology arms + SFFV Promotor + Turbo GFP and the other has LHA,RHA + SFFV Promotor + Turbo BFP. So that i can see homozygotes if a cell fluorescence in both colours. I do Cell sorting after 3 days (Trim58 file). After sorting the cells go in EPO medium which starts Erythropoiesis. In ca. 18 days the maturation process should be finished with most cell enucleated and 80-90% cells being Reticulocytes . I did a purity check on day 14 to see if my cells are still fluorescening (other 3 files). I saw almost no cells being positive if i use the same gating but i do see a lot of cells in between of negative and positive gates which i would think the cells are positive but less of the GFP/BFP is being produced because the nucleus is staring to condense and therefore the overall transcription is reduced. Less GFP/BFP, less strong signal. Does that makes sense?
I'm scared that there is some cell slippage when sorting and then the normal cells, without KO are proliferating faster as the KO cells which leads to less positive cells and making my experiment trash because the cells are still expressing TRIM58 so i can really make a conclusion what effects the TRIM58 has. Any ideas how to check how many of the cells in a population have the KO? I did DNA sequencing but here only the pieces are amplified that have the KO and don't give me any idea if in the population are normal cell too. I Have to little cells to make Western Blot to confirm if the gene is 100% turned off.
Any ideas how to make my KO efficiency better? I only have 0.2% of Homozygotes which leads to a too small cell population for me to do Western Blots and other analyses. On next experiment i'll use more AAVs(MOI=5000) and hopefully i get more Homozygotes.
Any ideas how to make my experiment better or any analyses i should do? All ideas and discussions are welcomed
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