| | Re: Generation of Knockout Mice
1.Clone targeting vector and design Southern blotting strategy
2. Introduce vector in embryonic stem cells (ESCs) and do drug selection
3. Identify correct clones by Southern Blot
4. (Optional) remove drug selection cassette from ESCs using Cre or Flp recombinase
5. Inject ESCs into donor blastocysts, transfer blastocysts into recipient female mice
6. Wait for pups to be born, hopefully they are mixed coat color (chimeras) indicating the ESCs contributed to the embryo
7. Breed chimeras and test if they transmit the mutation through the germ line
8. Analyze phenotype and publish in Nature!
Last edited by mmorgan; 11-23-2011 at 07:39 PM.