Gene not expressed in wild-type and knockout yet differences in microarray - (Dec/08/2010 )

How was the knock-out made? From what I read, I'm afraid your knock-out not only affected your gene of interest, but also has an effect on other genes. For instance, by deleting a part of the chromosome required for attachment to the nuclear lamina, you change the position of the chromosome in the nucleus, which can have an effect on the expression of other genes on that chromosome. Alternatively, you may have deleted an enhancer/repressor for another gene located some kilobases/megabases away from your gene of interest. Of course, this is all very hard to prove, but I would recommend checking the conservation of the intronic regions you deleted. If they are nicely conserved between different species, this may imply a functional role for these sequences...

Perhaps the genomic region deleted in your model does not code only for a mRNA, but also for a miRNA. A differential miRNA expression could clearly influence your microarray results as well as your phenotype (i.e., different cell growth).

dpo on Tue Dec 14 09:20:48 2010 said:

Hi dpo,

Two different knockouts were created by deletion of two separate exons. Your insights are perceptive. There is a gene that is transcribed in the reverse orientation to my gene of interest and they share a very short bidirectional promoter. Their expression patterns are quite similar in vivo. I've looked at the expression of this gene in my microarray data but unfortunately, it too is not expressed in both WT and KO. Your suggestions however now give me new leads to pursue.....I will see if any of those differentially expressed genes are located in or around the loci of my gene of interest. If your prediction is correct, it should show up in the data. I will look and keep you posted. Thanks for the great suggestions

96well on Tue Dec 14 13:50:12 2010 said:

Hi 96well,

Your suggestion is a great one too. I am now considering doing a microRNA microarray. I also recently learned about LINC - large intergenic non-coding RNAs. It shows how much remains to be discovered about gene regulation. A colleague of mine suggested another possibility......that the knockout effect was imprinted in the genome and passed on down the germline through methylation or acetylation of genes or chromosomes. This will be really tough to prove, although not impossible. Thanks for your thoughts

well, it is not that hard to prove if it is an epigenetic event, you can just go with bisulfide sequencing or chip for repressive histone marks (if it is MEFs the raw data is already public, you just have to download it and analyse the epigenetic marks for your gene).

The thing is, if it is not expressed in MEFs why analyse the KO in the first place? Is it expressed in other cell lines?
It is ok for it not to be expressed in wt MEFs, it is just weird the phenotipical differences, we do a bunch of KO mice in our lab and I never seen anything like that (except when certain areas in the 3'UTR or other regions involved in regulation got removed by mistake).

best regards
Radish

Hi Neuropath,

I am having exactly the same situation as you did 2 years ago. We have a knockout mouse, and I am seeing differences in the expression of a protein that is located in a different cromossome than the knockout gene. And this difference is also present in cells that do not express the gene that was knockout...
It would be great to know how your story turned out. Maybe it would help to give me some direction to what to do next...
Thanks a lot!