Hello every one,
I'm working on the in vitro expression regulation of a viral gene.
I'm looking at the splicing efficiency of several versions of the gene (comming from different genotypes). To do so I cloned the various versions of the gene in a pcDNA3.1 vector.
When expressed in eukaryotic cells, all vector expression but one give me the expected splicing pattern.
The odd one use a new splice site really close to the CDS start never reported in the litterature and absent in all other version of the gene.
The gene in the pcDNA3.1 is under the strong pCMV promoter which also add a 150ish base pair 5'UTR to the mRNA. Whereas, in vivo viral mRNA have a very short 5'UTR or none at all.
I was wondering if the 5'UTR addition and/or the strong expression could suffice to force this artefactual splicing ?
Thank you for your time !
Philippe.
Certainly, the length and sequence of the 5' untranslated region (5'UTR) can potentially influence alternative splicing, as changes in the 5'UTR can alter the accessibility of splicing regulatory elements, affect translation initiation through the Kozak sequence, and impact the secondary structure of the mRNA. Additionally, the use of a strong promoter like pCMV can lead to high gene expression levels, potentially influencing spliceosome assembly and kinetics of RNA processing. In your case, the observed splicing pattern in the pcDNA3.1 vector might be a consequence of the long 5'UTR and strong promoter, which could be introducing or disrupting splicing regulatory elements. Further experiments involving different promoters and 5'UTR modifications are necessary to elucidate the specific mechanisms underlying the observed splicing differences.
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