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Please advise on my transgene strategy :)


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4 replies to this topic

#1 Clare

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Posted 28 July 2010 - 05:01 AM

Hi everyone :D

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

#2 Rsm

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Posted 28 July 2010 - 06:34 AM

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
I got soul, but I'm not a soldier

#3 Clare

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Posted 28 July 2010 - 07:35 AM

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

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



#4 Rsm

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Posted 28 July 2010 - 10:44 AM

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? :P

Cheers,

Minna
I got soul, but I'm not a soldier

#5 Clare

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Posted 29 July 2010 - 12:46 AM

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

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? :P

Cheers,

Minna






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