twang11

Hi,

I am a struggling beginner in setting up my own luciferase experiment, and I am having difficulties making sense of the data.

I am trying to transduce a luciferase reporter into primary fibroblast cell lines. The lenitivirus also comes with a puromycin resistance gene. So, initially I would infect, select, perform an Alamar blue assay, and then lyse my cells to measure luciferase. I also quantified the total protein content in lysate. The general trends I got from luciferase/alamar blue and luciferase/total protein content is similar. However, I feel that my data is showing me that I have different transduction efficiencies across my different primary fibroblast cell lines. Ideally, I would like to co-transduce a reporter, like in cotransfection experiments, but I feel like a lentivirus based system is inherently different from transfection experiments where the transfection reagent being used to deliver the constructs is the same. So I have several questions as to why this is scientifically acceptable as normalization.

1. Is really every lentivirus construct that is replication deficient the same? Will buying a separate Renilla luciferase-puromycin lentivirus transduce the same as my reporter construct to actually use it effectively as an internal control?
2. If I select for my transduced cells again, I will be able to pick up cells that may only contain Renilla luciferase and cells that only contain my firefly luciferase, and would this serve as a good internal control across different cell types? Should I just not select for puromycin?
3. Is it possible that I can transduce one lentivirus over the other? Is there an optimal way to determine the ratios for experimental:control lentivirus?
3. What other careful considerations should I make if I co-transduce a second reporter as normalization?


Insight would be greatly appreciated!

Thanks