Why no splicing of vector construct?

Trei Martin gwmartin at mit.edu
Thu May 3 10:21:55 EST 2001



"remove the [nospam] Nicolas von Ahsen" wrote:
> 
> Hi all,
> 
> I am interested into the effect of a last intron on expression. I have
> therefore cloned approx. 50 bp upstream the splice donor site, the
> intron of interest, and approx. 50 bp downstream of the splice
> acceptor site in frame right behind the stop codon of the luciferase
> coding sequence (pGL3 vector).
> 
> The construct is as follows:
> 
> luc-gene incl. stop codon / my 50 bp in frame of exon DNA / splice
> donor / intron / splice acceptor / another 50 bp of exon / poly-A site

You should do a lit search of nonsense-mediated RNA decay.  I believe
that RNAs having introns downstream of a stop codon are recognized as
aberrant and are degraded.

What purpose is Luc serving in this system?

> I transiently transfect cells, isolate RNA, treat with DNAse.
> RT-PCR then shows that the intron was not removed by splicing.
> No RT control is negative.




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