Embryonic Stem Cell/Knockout Services

The facility offers a complete service from DNA preparation to screening of the newly genetically engineered mice.  The term ‘knockout’ is used here but the Core has just as often made ‘knock-in’ mice for our investigators and is experienced in the techniques of Cre/loxP conditional knockout mouse construction, including the desire to sometimes perform ‘secondary’ targeting events or transient Cre recombinase expression in homologous recombinant ES cell clones.  Most of our investigators design and construct their own targeting constructs, but for those who do not we can highly recommend trusted partners that can perform all DNA design and construction steps at relatively low cost.  Services offered by the Core include design and execution of PCR-based screening strategies, ES cell and mouse tail DNA preparation, ES cell gene targeting by homologous recombination, ES cell injection into blastocysts and chimeric mouse breeding.  Knockout mice are produced on a C57BL/6 background using our exclusive, published C57BL/6 LK1 embryonic stem (ES) cells.  Thus, knockout mice are produced on a C57BL/6 background, which is often the mouse background of choice for our investigators’ basic research.  Investigators are encouraged to submit a preliminary application form with the planned targeting before proceeding with targeting construct preparation.  Investigators are also encouraged to adhere to the DNA preparation guide.  Through this service, the core will generate chimeric mice by injecting ES cells containing site-specific genomic alterations (knockouts, knock-ins, etc) into normal blastocysts.  ES cells will be injected into a minimum of 100 blastocysts per homologous recombinant clone, and for up to 3 such clones if available.  The blastocysts will be transferred into the uterine horn of a 3-day pseudopregnant female and allowed to go to term (17 days after embryo transfer). Upon weaning 3 weeks after birth, chimeric mice will be separated and held until they reach sexual maturity (~6 weeks), then will be mated with a C57B/6 female up to 5 litters.

Due to the fact that this approach is time-consuming, it is recommended that certain guidelines be followed in order to minimize loss of time and reduce risk factors. These guidelines include:

• Before initiating the electroporation of targeted construct, all the procedural requirements in the application form must be met.
  
  
• The targeting gene detection strategies must be determined before any assigned electroporation day.  We HIGHLY RECOMMEND a Southern blot screening strategy but the Core itself can only offer to design and execute a PCR-based screening strategy, along with DNA preparation services if requested, for an additional fee.  We can however help those investigators in need of Southern blotting/probing assistance to identify potential partners for a ‘final’ Southern blot confirmation of positive results after initial PCR-based screening.
  
  
• A photocopy of the recombinant ES cell line screening results (Southern blot or PCR) should be submitted to the Core, in order to proceed with chimeric mouse generation.