I have KO cancer cell line, and I am applying shRNA to target some genes. My control is RFP-shRNA.
I ran western blot to check my protein of interest, and i have found that KO cell line with RFP/shRNA restore the phenotype of this protein of interest.
Does anyone has any explanation please ?
Dear Doaa
If I understood correctly, your cancer cell line transfected with the shRNA does not show reduction in protein levels right?
Could you please tell me how you deliverd the shRNA to the cells and if you confirmed transfection levels by Flow cytometry, since your shRNA is co-expressed with RFP (correct me if I got this wrong)?
In the event that you did confirm transfection efficiency and still the shRNA do not indicate reduction in levels of target protein, it is possible that the shRNA were not designed well enough to target a key region in the mRNA of the protein of interest (as in: maybe the region targeted does not exist in all mRNA isoforms for that protein)
How long are you measuring your protein expression after transfecting the shRNA? Transfections are transient so you need to measure within 24-72 hours post transfection.
Hi Aparajita Lahree & Sophie Sneddon
My problem is about the control shRNA cell line , I used RFP knockdown in my KO cancer cell as a control. This control cell acts like WT cell not KO cells.
The other shRNA KO cell lines where I target specific gene are working good, but not the control one.
for example:
in my KO cell line, gene X is lost, while shRNA control using the same KO cell line I found that gene X is restored.
any explanation please?
Hello again Doaa
Well, so this sounds like your control construct makes an already KO cell line behave like WT for the gene X. This is super strange. May I ask what the cell line is KO for and what gene X is?
Hi Doaa Glal,
1) The control KO is made with scrambled shRNA which does not target GeneX. So, If I am not wrong, the shRNA control should express intact geneX while the target should show lesser expression.
2) In other way, if you made Knock out cell line with stable RFP expression:
If you use shRNA for RFP as a control, and shRNA for Gene X; you should get downregulated RFP expression in control but not Gene X(It should be intact), and downregulation of GeneX in Test samples.
RFP expression is under different promoter (CMV, PGK1, TRE, etc), but shRNA is under independent promoter like U6. Both gets integrated to genome but express exclusively: RFP is for indication of stable transduction process.
If I am wrong, please correct me.
It is possible that you are control shRNA construct might express the gene which you knocked out in KO cells. Sequence the control shRNA construct and see. Otherwise, we need more information in terms of KO and shRNA construct and clone preparation.
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