I am trying to plan targeted gene knockdown in mammalian cell line but in my experiment the most critical issues are lasting and high efficiency gene knockdown. Which method is the best one to get gene knockdown with two features mentioned above?
Hello Mohammadreza,
I think there is not one perfect answer. You can certainly try Crispr or other gene disruption methods, however since shRNA-based suppression is been around for a while there are many reagents/protocols out there that you may find specifically for your cell line.
What cell line are you thinking of using? based on my experience with shRNA methods here is what I would recommend, although someone else may have a better idea with Crispr:
1. For long term suppression I would go with lentiviral-based transduction. I had T cells lines with >90% suppression for long periods of time (months.) The only reason I would toss them is because I wanted newer passes of cells. So what I would do is thaw a new batch of transduced cells. This lasting effect was observed with multiple different shRNAs.
Ofcourse, prior to all of this you need to find the most effective shRNA sequence, and probably would need to experiment with few of them. There may be studies out there that already published a good sequence for your specific gene.
2. You can always make a cell line with a complete gene knockout/disruption but that may take more time.
Hello Mahmood
Thanks for your suggestions.
I want to inactivate the genes involved in two pathways one by one to have the cell line with both pathway inactivated situation. But this inactivation will not be at the same time and maybe there is a gap time about two months between first and second inactivation.
I have never checked cell lines after one gene knockdown. is it reversible after several time passage if we use shRNA vectors to suppress the gene?
About using knockout/disruption technique you mentioned, i have to say that is right and in my situation is more difficult to prepare homozygote cell line because my cell line is polyploid.
In CRISPR we will have the same problem and on the other hand HR DNA pathway will try to repair DSB correctly.
I think the best method for me in this plan is shRNA method but i have to find the highest efficiency one.
I also think that shRNA is your best route at the moment. shRNA knockdowns should be stable as long as:
1. It is not a transient transfection such as electroporation or other methods. It needs to be stable via viral transduction. This is because as the cells divide the plasmid containing your shRNA it gets diluted down and eventually is lost with passes.
2. You keep a small amount of selective pressure via puromycin or other selective reagents.
As far as double knockdowns, I have made constructs in the past that can produce double knockdowns of two different proteins. However the placement of the promoter-shRNA constructs on the vector needs careful attention as if they are too close it can inhibit the expression of one of your shRNAs. I noticed this effect with U6 promoters on which the shRNA sitting on the 3' end was inhibited by the expression of the 5' shRNA. Overall though it can be done. There also may be other commercial vectors that are sold ready for the placement of two or more shRNAs.
Mahmood
Dear Mahmood
Thanks so much for your suggestions. i think i have to use viral based shRNA transfection like lentivirus. In this way i will have steady shRNA expression during subcultures.
The issue you mentioned about double expression of shRNA is important. Its obvious that you have more experiences than me. Do you have any document to help me to learn to design multi shRNA plasmid and help me to know what issues i should consider during this process.
I will ask about your suggestions and ideas about the probable problems i will encounter during my project.
Reza
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