Hello all, I want to immortalize human adult fibroblasts with a lentivirus plasmid containing hTERT under the promoter EF1A and puromycin resistance under the promotor mPGK. I performed a otimization transfection with a similar vector containing GFP and found 70% of transfection efficiency. After this step i performed the transfecton of the hTERT lentivirus with the same protocol. I also did a kill curve with the same cell lineage without transfection and found that 1 ug/ml of puromycin killed all cells after 8 days. After transfection I tryed to select the hTERT fibroblasts with the same concentration of puromycyn, but all cells died in the next day. I Tried again with 0.5 ug/ml but all the cells died again. I did a PCR that confirmed that the hTERTB plasmid was present in the cells, so they should be puromycyn resistant. I suspect that the presence of the plasmid coulb be toxic for the cells because the not-resistant control cell supports the same concentration of puromycin for longer times than the resistant.
In this situation, I would first recommend adding additional amino acids to the cell culture.
I guess they're stressed out by the electroporation. You're doing it right. Try additional lower concentrations on the real transfected culture, until you find one where there is survival. These will likely be enriched for your integration. (if your plasmid works at all...)
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