Dear all I am planning to transiently overexpress a proten (PROTEIN A) in a HEK293FS cell line, in the same experiment I want to knockdown another protein (PROTEIN B) in the same cell line that transiently overexpress(PROTEIN A) in order to know whether knocking down of Protein B will decrease or increase Protein A.
However, the problem is that the cell line becomes confluent within 3 days, but it needs 2 days to overexpress the Protein A and 3 days to knockdown Protein B. So how should I design such experiment in a proper way?
Does anyone has experience in doing such kind of experiment? any suggestions are welcome Thank you.
Hey,
Although I do agree with Jiang that you can generate a stable cell line with a knocked out gene, and start from that point to overexpress the gene of interest. However, I believe that you can simply cotransfect your HEK293 cell line with the Plasmid DNA (of the gene to be overexpressed) and RNAi (to knock down the other gene) at the same time.
For further details please refer to the below link to a protocol for cotransfecting HEK293 cell line:
https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.43693.file.dat/cotransfecting-rnai-dna-lf2k-web.pdf
Regarding the design of the experiment, I think you will need a negative control; cells treated with the RNAi (knock down) only; cells treated with the gene wanted to be overexpressed only; and finally the cells cotransfected with both the RNAi and the Gene to be overexpressed.
Good luck
Hi,
I would like to know that, Is it necessary to do BOTH overexpression and silencing of a gene in a study?
Actually, when I am overexpressing a gene, after that my treatment reducing the expression of the gene while I am trying to silence the gene to understand the process in another way round i.e, assuming silencing of the gene, and then treatment could reverse the effect of silencing and restore the expression. But the problem is upon silencing, cells start dying.
So, in this situation, do ONLY the results of the overexpression part will it be acceptable?
Regards
Saurabh
Saurabh Mandal your explanation of the question is hard to understand. what is the treatment (over expression or knockdown)? If your cell are below 80%, during the transfection the resultant cell death will reduce the number of cells below the level from where the revival is very difficult. Furthermore, if you are using any antibiotic during the transfection, it will also add up to the death. Very high passage number could also be a problem.
Choudhary S Ahmed First, confluency and passage number was good enough for transfection. No antibiotic is being used during transfection.
Now let make it easy to understand, "to study any particular gene role or function in cells, do we need to do both overexpression and silencing of that gene?" Only overexpressing and studying things won't be justified.
Regards
Saurabh
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