I have constructed and transformed my gus construct in the following way
construct : gene promotor-5utr-genomic seq(introns included)-3utr----gus . My gene produce two kind of transcripts. So i want to know the spacio-temporal expression of both transcripts in arabidopsis thaliana. I have done the gus stainning but did not get any gus activity in positive tranformants.
Please tell me does gus contruct work with genomic sequence or only with CDS?
Hi, Sehrish.
I've constructed promoter-gene-gus (including introns), from genomic DNA and expressed in A. thaliana with no problems with gus expression for like 15 genes, so I don't think there's a problem with that. Sometimes introns contain regulatory information, so I think is even better for reproducing expression patterns. I can only think of the following:
a. You included the stop codon of your gene (translation will stop there and never get to the GUS, the transcript may even be degraded because of non-sense decay.
b. Your gene and GUS are not in the same reading frame, have you checked this in the expression vector? I usually sanger through the junction when using new vectors.
c. Your promoter is not driving the expression, maybe you used a promoter insufficient to reproduce expression, and/or your promoter does not actually drive expression in the tissues you're checking.
Best of luck!
JA.
Hi, thanks JA
I have mutated the stop codon of my gene and Gus gene is also in the same reading frame.
I have also constructed the Promotor-CDS-GUS that plants are giving good results after staining at four leave stage but I don't know whats the problem with the genomic construct. Ok I will try these with different tissues aswell, thanks
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