Baf3 cells were transduced with a mutant gene and now I want to silence this gene. Does it make sense to silence a transduced gene or do I have to look for a cell line that naturally has the mutation?
Hi Yasir,
To transduce the IL3 dependent Ba/F3 cell line with a mutant gene and then to try to silence the same gene seems a bit counter intuitive, as this in reality should bring you back to wild type Ba/F3 cells (or one at least transduced with empty vector only). So any difference in Ba/F3_empty vector vs Ba/F3 mutant is a valid functional comparison.
We also work with Ba/F3 cells to test if our mutant constructs can transform the cells to IL3 independent growth. An ability often related to oncogenic driver mutations - mostly in kinases.
What we sometimes need to do is silence any endogenously expressed gene if the the phenotype is not strong. You can design shRNA/siRNA to the mouse orthologue which should silence this but not the mutant variant should it be of human origin and have no homology in the region targeted.
Of course, finding a human cell line that has the mutation directly can be useful to determine the function, but not always, as immortalized cell line often have a raft of mutations which may affect your read out (what ever that may be).. SO the Ba/F3 system is "cleaner" in that it will only have your mutation present.
I hope this helps, All the best with your work.
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