retrovirus infected 3T3-L1 cannot differentiate and die

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nailone
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retrovirus infected 3T3-L1 cannot differentiate and die

Hello,
I met with a problem that when infected with retrovirus, my 3T3-L1 cells cannot be differentiated and died a few days after differentiation medium induction. Before infection, 3T3-L1 cells grew healthy and can be differentiated.

I started differentiation 2 days post 100% confluence, used differentiation medium containing 5ug/ml insulin, 1uM DEX and 0.5mM IBMX for the first 2 days, shifted to medium containing 5ug/ml insulin in the following days and refreshed medium every 2 days. Cells started to die about 3-5 days after induction and no differentiated cells could be found.

I used pLkO.1 retrovirus vector for siRNA knock-down.

I tried different virus titers but got the same result.

Would you please help me with this problem? Many thanks.

Jason King
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 Did you have a control

 Did you have a control infection ie. the same retroviral vector used to transduce the cells with a control siRNA? If not then you don't know whether the gene knock down killed your cells or something linked to the viral infection.

just an observation : it's unusual to be using a retrovirus for shRNA knockdowns these days. Doesn't everyone use lenti vectors now?

Did you use anything potentially toxic to promote infection (eg. Polybrene)? Again, if this was a problem it would have been picked up by the control experiment.

what was the shRNA supposed to be knocking down?

nailone
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Thank you parvoman.

Thank you parvoman.

The control shRNA group also cause the cell death.

Sorry, pLKO.1 is a lentivirus vector. I only know that lentivirus can infect non-dividing cells unlike retrovirus but what's the difference with retrovirus for 3T3-L1 cells.

I did not add anything else to promote infection.

The target of the shRNA is a protein coding gene.