Unable to get Puromycin resistant colonies following Lentiviral transduction

We are developing _Med23 _knockout cells. We successfully cloned Med23 sequences (gRNA coding cDNA, 4 different targets) into pLentiCRISPR v2 to generate pLentiCRISPR v2.Med23.

Thereafter, we produced Lentiviral vector by transfecting pLentiCRISPR v2.Med23, pCMV delta R8.2 and pVSV constructs in HEK 293T cells. These Lentiviral vectors were transduced in HeLa cells in presence of polybrene (2 ug/ml) for 48 h and then selected by puromycin (15 ug/ml) for next 48 h. However, all the cells died following puromycin treatment.

In a control Lentiviral vector reaction where pLentiCRISPR v2.Med23 was replaced with pHR-CMV-GFP, the pesudotyped viruses produced GFP in >80% transduced cells suggesting lentiviral production was OK.

Can you please help me obtain right puromycin-resistant clones?


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Bronze Ghoulalmost 5 years ago

Have you optimized your puromycin concentrations? It seems really excessive to me. We usually just use 1-2 ug/mL.

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