Transgenic construction to study a promoter - (Nov/14/2006 )
I'm trying to study the regulatory sequences of a specific promoter.
So I will fuse the promoter to a LacZ reporter to generate a transgenic mouse and see If It can drive expression of the LacZ in specifics areas of the mouse brain.
I'm just wondering If in my construction I have to include the ATG (translation start site) of the gene's promoter, or If it will works only with the transcription start site, knowing that the LacZ Reporter has a start codon?
Thank's for your help!
Including an ATG can screw up your construct if the reading frame is shifted. I would strongly advise against this. Sometimes you can include 5' untranslated regions of your gene (up to the start codon) if you believe there are regulatory regions present in this region. Also, before you do your mouse work, have someone look over your construct. It can save you about a year of your life in case you have made any other errors.
Thank you so much for your help.
I will do my construction including only a part of the 5' UTR and not the ATG start codon.
I have another question, if I add the Lacz reporter gene including its ATG start codon, do I have to put it in frame with the promoter sequence, or it doesn't matter?
I supposed that it's not necessary to put the promoter and the lacz sequences in frame?
There's is really no "reading frame" established until you initiate translation. It may help to put a kozak sequence upstream of lacZ, but it is not always necessary.