I transformed an Arabidopsis to express miRNA that targets a gene family with conserved sequence, under a root directed promoter. I found that in the roots (in the leaves there is no change) of the transgenic plant there is an increase on 2-5 fond of the targets transcripts using RTPCR, compared to WT. I haven't checked protein level or activate level yet.
Could it be that there is a feedback loop that induces production of more transcripts of these enzymes because their activate is low?
has this been recorded before?
Dear Zhang:
I don't think i understand what you mean.
The sequence was generated using WMD3, and BLASTed for off tragets. Then synthesised by MERCK, then sequenced several times in different constructs by the Weizmann institute sequencing service.
And post transformation I used BASTA selection on the same construct, and PCRd the miRNA fragment out and the size of the fragment matches expectations. Haven't sequenced it from the plants, but it should be the same.
Hi Chen,
I have experienced something similar with an RNAi line I received from another lab. We consistently measured enhanced mRNA levels instead of reduced. I never found out the reason but managed to get another mutant fortunately.
Is this response consistent in multiple independent transformed plants (off target effects)?
At the time i read somewhere that miRNA not always lead to degradation of the target mRNA but may stall translation at the ribosome instead, leading to accumulation of the target mRNA. If it is crucial to your further work, I would test protein levels (you might wanted to do that anyway for your WT responses?).
Also check out this paper, which I guess is not limited to animal cells: https://www.pnas.org/content/101/7/1892.long
Good luck!
Hey Sjon
I have also read that there is rarely change in mRNA transcript levels in microRNA transgenic plants. And microRNA inhibits gene expression vie both translation and degradation of mRNA transcripts.
This exists in the two independent homozygous lines I have.
I have not done a full transcriptome, but have not seen changes in 3 other genes I have checked so far.
I’m not sure that genes that change cell cycle in cancer calls are as controlled as we’d like to think, although showing that different cited siRNAs can cause an increase of transcription is interesting, thanks.
I’ll have to check protean and activity levels.
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