Hi all,
I am struggling for sometime to understand how rescue works. In my case, I kd (knockdown) N-WASP (actin regulating protein) in HaCaT cells using shRNA based Lentivirus mediated transduction.I get almost 95-100% knockdown on western. These cells have significantly reduced proliferation compared to empty Vector (Control). ---> So I say Kd of this protein, reduces proliferation.
So Now I try to rescue the phenotype using shRNA resistant construct of my gene on these kd cells. I get back around 60% protein expression in western. But my phenotype is further reduction in proliferation.
In summary, Kd causes--> reduced proliferation and rescue --> reduces proliferation even further compared to kd.
Does someone has any idea about how rescue works? Pls share your suggestions and any papers that might help to explain these results.
Thanks
Apoorva
How do you measure proliferation rate?
I want to know more how far does it further decrease the proliferation rate?
How can I rescue the phenotype of my gene after knockdown using shRNA (to show the specificity of the shRNA)?
You can rescue the phenotype of the gene by over-expression of a cDNA or introducing large amounts of protein into the cell to out-compete the knockdown.
http://dharmacon.gelifesciences.com/resources/faqs/how-rescue-phenotype-gene-knockdown-using-shrna-show-specificity-shrna/
If you see the same effect in rescue experiment as you see in Wt cells upon KD of your protein of interest it most probably means that it is OFF target effect (something else was targeted by your shRNA along with N-WASP RNA).
One type of rescue mechanism is the emergence of second-site revertants. But that usually works only for single amino-acid mutations. For knockdown (which I assume is the same as a knockout), I am not not sure ..... Perhaps gene amplification of another protein that is functionally homologous to the protein gene originally knocked out is occurring.
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