retrovirus infected 3T3-L1 cannot differentiate and die

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nailone's picture
retrovirus infected 3T3-L1 cannot differentiate and die

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
Jason King's picture
 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's picture
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.