Inducible Gene Targeting in Mice Using the Cre/lox System.

[Kyle Legate]

S. Ottawa Grad Student wrote:
> Hello Everyone,
> 
> I am new today to this group. I have looked at the previous postings I
> could find regarding creating
> KO mice using the Cre/Lox System and none of them seem to give me what
> I need.
> 
> What I would like is a detailed, specific protocol for achieving this.
> I have not been able to find such a document with sufficient technical
> information regarding procedures, possible snags and useful tips on
> troubleshooting techniques.
> 
> If anyone has such documents or knows where I can obtain them please
> let me know.
> 
For a good step-by-step guide to the embryonic stem cell work, you 
should try to get a copy of "Integrin gene targeting" by Talts et al:

http://tinyurl.com/2kakzu

Additional information can be found in Manipulating the Mouse Embryo, by 
Nagy et al. As for construction of the vector, I don't know of any 
better way than to sit down with someone who has done it before. It's 
easy to make a stupid mistake at this point that can cost months of time 
(loxP sites in opposite orientations, for example). Perhaps if you join 
the transgenic listserv and ask your question there someone will have 
some literature to recommend.

What you can begin immediately, while you gather additional information 
about how to construct the vector, is to screen a commercial genomic 
library using a fragment of your gene of interest as a Southern probe. 
The result will tell you which PAC or BAC clone to obtain from the 
company to use as starting material to make your targeting construct. 
You will need around 20 kb for both recombination arms, and they must 
have no mutations (lest you lose most of your targeting efficiency) so I 
wouldn't   even consider trying to obtain it through in-house PCR.