I want to knock down CSF-1 (M-CSF) in H358 human lung cancer cells. I bought shRNA from Sigma, a validated one, and checked that it has a seeding sequence in all 4 splicing variants of the CSF gene. There was a very good decrease in CSF RNA, about 80%. About a month after, the level of CSF RNA started to elevate, addition of puromycin caused some cell death, but the survivors show the same high CSF level. Why? What am I missing?
What role plays CSF-1 for the cells? Depending on how important it is, you put quite some selective pressure on the cells to find a way around this knockdown.
For knockdown experiments I usually try to keep the cells as short as possible under knock-down conditions to avoid this. Depending on the target I also use the selection markers.
Hi Lena..so you make stable clones, expressing the shRNA? did you keep always in selection medium? how many passages they have before the RNA levels starts to rise again? Usually it is better to keep shRNA clones always in selection and to not use more than 6-8 passages (depending on doubling time of cells)...
you have to do a QPCR to see the plasmid integration in your cells to be sure that is a stable clone.
Potentially, there could have been.a negative feedback loop controlling the expression of CSF-1 gene...resulting from low CSF or incomplete KD of CSF-1. It happens with KD of c-Fos with antisense oligo to c-fos mRNA.
If they are stable cell lines you might have selected a clone which has a mutated target site. Did you sequence the target sites of your cell line?
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