Hi, guys, I want to study two genes' function in endothelial cells at the same time. I would like to use siRNA to knock down one gene and lentivirus to overexpress another gene. Do you have any suggestions or established protocols that I can follow? Another issue is that I want to use these cells for glycolysis seahorse assay later, should I do the siRNA first then overexpression or overexpression prior to siRNA knockdown, because the microplate will be used. Thanks in advance!
Hi Liu
You could express an shRNA cassette from the lentivirus itself, as well as the gene to be overexpressed. This has been carried out extensively (eg https://stm.sciencemag.org/content/8/342/342ra79?rss=1). It would ensure that knockdown and overexpression are happening in the same cells.
In terms of your question relating to timing of knockdown, you could use an inducible promoter to control when the shRNA is expressed. This is achievable if you encase the shRNA in a miRNA scaffold (eg https://www.thermofisher.com/order/catalog/product/K493900#/K493900), to exploit Pol II processing and regulatory function. You can then switch on the shRNA component when optimal for your metabolic analyses.
Best wishes
John
Hi, John Counsell
Thanks for your answer. Given that we will perform the glycolysis seahorse assay in a 96-well microplate, so we are planning to knock down one gene first and incubate these transfected cells with lentivirus to overexpress another gene. How do you think about this plan?
In principle that should work fine, I’ve done similar before. Obviously make sure to run appropriate controls to monitor possible effects of each treatment on the other. This ultimately depends on which gene you are knocking down and which you are over expressing.
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