Generation of Transgenic Mice

Transgenic mice are produced by microinjection of DNA into the pronuclei of fertilized single-cell mouse embryos. Transgene integration is random, with multiple copies of the transgene typically integrating into a single chromosomal locus in the embryo.

If integration takes place prior to the first nuclear division, then all cells in the founder animal will carry the transgene. Potential founders are then genotyped to confirm the presence of the transgene.

Procedure: The Transgenic Mouse Core Facility staff microinjects a minimum of 200 fertilized embryos from the matings of outbred B6D2F1 mice or from the mating of inbred C57BL/6N mice, implants the microinjected embryos into 10-15 pseudopregnant recipients for each transgene, and takes care of pregnant females and their litters for up to 2 weeks after the pups are born. At this time the pseudopregnant mothers and their pups are transferred to the investigator.

If all project criteria are met, the Transgenic Mouse Core Facility will guarantee three founder animals for each transgene. However, it is impossible to predict whether the transgene will be expressed in the mouse. For this reason, the Transgenic Mouse Core Facility cannot guarantee expression of any transgene provided by an investigator.

The fee for this procedure is $3,600 for B6D2F1 mice, and $4,600 for C57BL/6N mice.

Transgenic Construct Criteria

• 25µL of transgene DNA sample at a minimum concentration of 50 ng/µL in Microinjection Buffer (10mM Tris, pH 7.4; 0.25mM EDTA) must be provided by the investigator. EluTip-D (VWR Catalog # 28151-080) is recommended for purification.
• The DNA solution must have an OD 260/280 ratio of 1.7 or better.
  A gel photograph of the DNA fragment and its parental construct showing good separation of the transgene DNA from its vector must be provided.
• A linear map of the construct showing restriction sites used to release the transgene DNA fragment must be provided.
  If the transgene is from a BAC, P1 or YAC clone, please discuss protocols for DNA preparation with our Core staff.

Scheduling Requirements

Services cannot be scheduled until all project criteria are met and a Pronuclear Microinjection Request Form approved.

• Prior to the initiation of any pronuclear microinjection service, all investigators are required to have a proven screening strategy, which could be Southern blotting or PCR. A serial dilution of the transgene can be used to test the PCR sensitivity.
• An approved, active animal protocol justifying the particular experiment is also required. The Transgenic Mouse Core Facility will not initiate any service without a valid animal use protocol number. (Visit the Animal Care and Use Committee website for more information about protocol numbers).
• At least 14 days advance notice will be required for the cancellation of a project; otherwise a cancellation fee will be charged to cover any costs incurred due to the cancellation (e.g., for donor mice already purchased in anticipation of the project.
• Services will be provided on a first-come, first-served basis, and a tentative date for initiation of services will be provided upon the approval of the appropriate procedure request form. (See Procedure Request Forms page for additional information).

Additional Information: These Cell Migration Consortium/Nature Publishing Group websites provide useful background or resource material for those unfamiliar with the procedures: Beginners with Transgenic or Knockout Mice, start here. For a guide to the basics of preparing Gene Targeting Constructs, go here. Note: If you cannot make your own gene targeting constructs, there are companies that will make gene targeting constructs for a fee. The Transgenic Mouse Core staff have not had direct experience with any of these companies, but would be happy to provide investigators with their names. Call Dr. Liqing Yu at 716-0920 for more information. ES Cell Lines Available for Generating Some Knockout Mice Some knockout mice can be made without the investigator having to generate the targeting constructs or the targeted mouse embryonic cells.  Please check the following links to see if the gene of interest has been targeted in the mouse embryonic stem cells or even in mice. The International Mouse Knockout Consortium: National Institutes of Health Knockout Mouse Project (KOMP) European Conditional Mouse Mutagenesis Program (EUCOMM) North American Conditional Mouse Mutagenesis Project (NorCOMM) Other Useful Weblinks: International Gene Trap Consortium The Mouse Genome Informatics Mutant Mouse Regional Resource Centers International Mouse Strain Resource (IMSR) Texas Institute for Genomic Medicine (TIGM) Call the Transgenic Mouse Core Facility's director, Dr. Liqing Yu, at 716-0920 for more information.