u/Status_Constant_8519

Hi everyone,

I’m having trouble with lentiviral transduction in mouse c-Kit⁺ hematopoietic stem/progenitor cells targeting Dnmt3a. I designed three shRNAs (pLKO); one shows strong knockdown in NIH3T3 cells. However, in c-Kit⁺ cells, shControl gives ~70% transduction, while shDnmt3a drops to ~7% after 10 days, despite similar MOIs(through gfp tagged in plko backbone)

This suggests possible toxicity or off-target effects. The other two shRNAs show weaker knockdown (5–20%) in qpcr but normal survival in c-Kit⁺ cells. I do not even trust that this because it very small knockdown

Any advice or similar experience would be appreciated. (shRNAs designed using Thermo Fisher BLOCK-iT.)