Please advise on my transgene strategy :) - Thanks! (Jul/28/2010 )

Hi everyone

After a wonderful holiday in Germany, I have finally left Cambridge, UK behind and I'm now in Edinburgh! wOOt!

Anyway, I just wanted to ask some advice about my construct to make a transgenic mouse and/or rat.

Basically I want to put in: promoter/GFP/important intronic element into a lentiviral vector (and go on to infect embryos with the virus). I have been reading that intron splicing seems to play an important role in the success of going germline. Now from what I can gather, most people seem to use a cDNA sequence in their construct (usually their gene of interest?). But in my case, I guess the only real cDNA is from GFP - so would I put an intron and splice donor/acceptor sites within the GFP??!!

Thanks in advance for your help!!!!

Clare

That depends on what you want to find out... If you're about the function of your intron as an enhancer, I guess you'll put something like minimal promoter - intron - eGFP.
However, before you start your mouse work, I would really recommend to test your construct in cell culture. Then you can design your reporter much better and quicker. Secondly: Please don't do transgenics! You'll have random multiple integration and site-of-integration effects. This will cost you much more time than a proper targeted integration (e.g. into the Rosa26 locus).

Cheers,

Minna

Hi Minna,

We already know the function of the promoter and intronic element and so the reason why I have been asked to do this is just to make a rat version of the existing mouse. The whole construct used to make the mouse version is too big for lentiviral work, so I am essentially making a cut-down version.
Clare

Minna on Wed Jul 28 14:34:35 2010 said:

That's good to know. Sometimes you find people being a bit too quick, sorry that I took you for one of them.
Anyway, no reason not to make a knock-in rat . Wasn't there a recent paper about rat ES cells with germline transmission? They were from Edinburgh and Cambridge, weren't they?

Cheers,

Minna

no probs, I can be quick to judge too!
Yes, those recent papers about rat ES cells were from Edinburgh/Cambridge I didn't work on ES cells in Cambridge though.
Clare

Minna on Wed Jul 28 18:44:04 2010 said:

That's good to know. Sometimes you find people being a bit too quick, sorry that I took you for one of them.
Anyway, no reason not to make a knock-in rat . Wasn't there a recent paper about rat ES cells with germline transmission? They were from Edinburgh and Cambridge, weren't they?

Cheers,

Minna