When generating a transgene with an artificial 3’UTR, is it sufficient to have a poly(A) signal at the end
or is it necessary (or better) to also incorporate a characterized 3’UTR known to have a transcription termination site? (like unc-54 3’UTR)
Also, are there any known transgenes have been tested & characterized by northern? how does the RNA size correlate with the transgene?
I came across the following paper:
Haenni et al. Nucleic Acids Res. Nov 2009; 37(20): 6723–6736.
Regulation of transcription termination in the nematode Caenorhabditis elegans
“By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites
into the 3′ flanking regions of the nematode genes before it terminates.”
So I would expect that the RNA would be ~1kb more than the transgene.
I thought I would keep this question warm as it has already dropped off the first page.
This is not really my area (so please, other members working in this area intevene here). But, to your first question, if I remember correctly as long as the 3’-UTR doesn’t contain a stronger PAS upstream of the one you want to add and also has the required downstream and upstream elements (DSE/USE), then you should get efficient 3’ termination (Nick P., my apologies if that’s too simplified). the advantages of the characterised 3’-UTRs is just that…they are already characterised.
"By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites
into the 3′ flanking regions of the nematode genes before it terminates."
So I would expect that the RNA would be ~1kb more than the transgene.
Isn’t it the case that once Pol II terminates transcription and there is 3’ cleavage/release of the mRNA, then although the transcript length is ‘fixed’ it is still subject to regulation (in terms of how stable it is and how efficiently it is translated) by 3’-UTR sequences? Hence, whilst the longer transcript might be detected on a Northern, 3’-UTR sequences in these ‘long’ mRNAs might bind regulatory elements such as microRNAs or similar that influence mRNA stability. What you see is not always what you end up getting…