Transcripts analysis

[roniadam]

Hi

I am working on in vitro expression analysis. I have transfected a mammalian cell line with a gene of wild type and mutant one. surprisingly, I found that the transcript level of the wild type gene was less than the mutant (which is against the logic theory where the wild type should be expressed more than the mutant)..... I wondered what is the reason behind the less expression of the wild type than the mutant one.... any idea because I got exhausted with this without finding an answer ????????

[JackBean]

what promotor did you use? What gene was that?

[kolean]

What was the vector you used? Are they both the same? and how was the mutant made? is it deletions or substitutions?
Was the one transgene integrated into the genome perhaps?
How did you quantify the transcripts? perhaps the vector was not being being replicated thru the cell cycle?
My favorite of course would be epigenetics. How is your gene normal expressed? did you add some cis acting elements to your vector with the gene? does the mutation confer some kind of cis acting element?

[roniadam]

I used T-A cloning vector and for the promotor, I used the gene's itself promotor and it showed a nice expression with the mutant but less expression with the wild type. the mutation was a point mutation (single nucleotide substitusion). Any idea for how to make the wild type to be more expressed than the mutant one??? or any idea about the causes of this less expression. ????

[JackBean]

My guess would be that the WT is expressed as normally and as is it active, it is not needed much to express, but the mutant is lacking activity and thus must be expressed in higher amounts