By retroviral over-expression system I couldn't get the same amount of over-expression efficiency (increased expression in protein level as detected by western blot).
I tried overexpressing a gene by retroviral system, at the first time I could get a good amount of protein increase and mRNA increase but the second time I couldn't get the same result. There are different things I did while performing the protocol, what is the most possible cause?
1. Different passage cells, the first one is 19 passage, and 2nd is 46 passage
2. Different cell density for cell harvesting (lysate preparation for western blot)
3. The first one I transfected 2x, while the 2nd one I transfected 1x. The assistant professor said that the amount of transfection will not influence the increase/decrease gene expression (?)
Your feedback is very much appreciated.
I would assume it's the cell passage. 46 is a pretty high passage number and cell aging / scenescence might reduce their ability to express your protein.
Perhaps try to keep all the conditions the same for future experiments (confluence, protocol). And not use cells after passage 25-30 to achieve a better reproducibility.
All the explanations are valid, point 3 being potentially the most relevant. If you transduced the cell twice with your retroviruses, this can explain the high overexpression levels observed in the first experiment. You have basically used at least twice the MOI used for experiment 2. Retroviruses are integrating into the genome and the levels of overexpression, at least up to a certain point, are a function of the integration events. Other factors will influence the expression of your transgene, as the integration sites and promoter silencing over time... but overall the MOI is a good starting point to start troubleshooting your problem in my opinion.
Francesco Aulicino if I repeat transduction/infection of target cells to 2x, will it affect the off-target effect of the plasmid? Is it wise to do so? I tried again with increased cell confluency 1x transduction/infection and result is better. I am a fairly beginner, I just realized that cell confluency really affect the expression of this protein. At the beginning I never pay attention to that and directly make protein lysate from subconfluent or even low confluence cells. what a sad thing
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