5 replies to this topic

[Cell Grower]

Hi everyone,

I'm currently trying to knock-down the expression of the FCGRT gene (encoding the neonatal FcRn recpetor for IgG) in liver cells, so I chose to tansfect them with some different shRNA-encoding plasmids provided by a supplier.
Since there is no efficient antibody marketed, I can't possibly check the diminution of the receptor expression, so I tried to check it at the mRNA level by RT-qPCR.
The problem is that, compared to wild-type cells, the mRNA level of FCGRT transcripts is higher in cells transfected with the shRNA control. Is it possible that the introduction of the irrelevant shRNA in those liver cell induced an upregulation of the gene ?
However, I managed to obtain some cells that seem to be knocked-down for the FcRn (still at the mRNA level), compared to shRNA control-cells.
My question is, do you think it is relevant to continue and try functionnal experiments on those FcRn-KO-cells compared to the shRNA control-cells, since the level of transcripts supposed to encode the FcRn receptor is higher in it than in wild-type cells ?

Thanks a lot !

[pcrman]

I believe there must be something wrong in your system: control shRNA, plasmid preparation, transfection efficiency, PCR system etc. I also believe that the shRNA you obtained from the vendor should be validated and known to work. If you don't even get decent mRNA knockdown, there is no use of going further.

[Cell Grower]

Thanks for your answer,

I don't believe the problem is due to what you pointed out. In fact, the transfection efficiency was controlled (and cells were selected by their ability to resist under a certain dose of Puromycine, which resistance is given by the shRNA plasmids) as well the plasmid preparations. The PCR system, I do agree is not the best way to control wether there is an expression or not, but I can't do a WB since I have no efficient antibody against FcRn.
Moreover, the vendor ensure at least a 70% mRNA knock-down. So I manage to have some good FcRn-KO-cells compared to control cells tranfected with control shRNA, but the real problem is still that those control cells seem to express the FcRn receptor more than wild-type cells, and I don't know what it is due to ?

[vitalgene]

how about siliencing upstream gene to the gene of your interest. just guessing !

[zodiac1505]

I have had that happen to me once. My gene of interest was knocked down at the protein level but I saw an increased expression at the mRNA level. One of the members mentioned then that it could happen if your gene of interest has a feedback loop. Suppose your gene controls its own expression through a negative feedback loop and once the protein level goes down, the gene compensates by transcribing more mRNA and hence the enhanced mRNA expression. It turned out to be true in my case. The other thing could be if your gene has isoforms and the shRNA is knocking down one of the isoforms and compensatory mechanisms lead to an increase in the others.

[pcrman]

This phenomenon can now probably be explained by the RNAa mechanism. In C. elegans, 22G-RNAs (which are secondary RNAs derived from piRNAs) antisense to endogenous mRNA are loaded by the CSR-1 Argonute protein to form a complex which then enters the nucleus. the 22G-RNA/CSR-1 complex binds to nascent mRNA to promote the transcription of mRNA epigenetically. You can read the following papers:

 

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

Seth M, Shirayama M, Gu W, Ishidate T, Conte D Jr, Mello CC.

 

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

Cecere G, Hoersch S, O'Keeffe S, Sachidanandam R, Grishok A.