Need help designing a construct to generate a transgenic mouse - (Aug/22/2006 )

Dear all,

This is the first time I have to design a expression construct to generate a transgenic mouse and I need a bit of help !

Basically I need to generate a mouse that expresses Dlp1 in a tissue specific manner. Since I'm going to have to assess the effects of the expression of this protein in several tissues I was thinking of making a construct with a ubiquitous promoter (CMV ?) followed by a STOP codon between two LoxP sites, then the cDNA followed by a poly A stretch (BGHpA).

However, I was told that when a mouse harbouring repeqted insertions of this construct was to be crossed with a Cre mouse, recombination would occur not only between the two LoxP sites arround the STOP codon but also between LoxP sites in adjacent expression cassettes. This would mean that I would most probably be left with only one copy of my cDNA being expressed .... which I don't think is going to be enough.

To go around that potential problem I thought of designing a contruct for the expression of a polycistronic transcript which would look like this

Promoter (CMV???)----LoxP-STOP-LoxP----cDNA--IRES--cDNA--IRES--cDNA--BGHpA

Do you think that this could work ? Will there not ne recombinations between the different cDNA sequences and excision of a region of the construct ? Is CMV a good choice of promoter ?

I thank you all for your patience in reading this and for your help.

All the best


P

From my limited knowledge, your origional idea might work. Some people end up using 3 lox sites for generating targeting construct for knockin (lox-homologous dna-lox-neo-lox). Then remove the neo by closing with cre mice. Some mice will be (lox-dna-lox) some will be (lox-neo-lox) and some will be (lox-lox -> no homologous dna or neo resistance). You would just need to screen the pups (but you would need to screen more pups than usual).

Another approach would be to use a double transgenic approach, such as tet-on or tet-off.

Or if you know of a tissue specific promotor, then you can use that instead of CMV in a single transgenic mouse.

Good luck and happy cloning.

Thanks for your reply. Would you by any chance know of good promoters for ubiquitous expression of a protein in mice, something like a beta actin promoter. Do you know of any other ?

All the best

P