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The Tulane Transgenic Mouse Facility |
Knockout Mouse Production by ES Cell Injection
The "loss-of-function" approach to studying gene function in mice has been very successful in recent years. The most commonly used and most informative "loss-of-function" approach is to study the consequence of eliminating a gene in the mouse. This technique, termed gene "knockout", has been well established in the mouse. Analysis of the "knockout" mouse phenotype can allow us to deduce the function of this gene in a biological process. In addition, it offers a genetic toll or model to study complex genetic pathways during development or disease processes.
The most crucial step in creating a knockout mouse is the preparation of the knockout embryonic stem (ES) cell line. This process utilizes homologous recombination to target and replace the endogenous gene with a modified nonfunctional copy. These targeted ES cells are injected into the blastocysts of donor mice. The injected blastocysts are then transferred to the uterus of a pseudopregnant foster mouse. The injected ES cells co-mingle with the ES cells of the inner cell mass of the donor blastocyst. Thus the resulting pup is chimeric, with both the injected and donor cells contributing to the structure of the mouse. Chimeric mice are easily identified by coat color, as the chimeric mouse will have bands of fur displaying the colors of the background donor and ES cells.
To be provided by investigator
- Completed Request for Service form
- Targeted ES cells cultures that have beeen tested for mycoplasmas and found clear (The investigator is responsible for getting approval from the Institutional Biosafety Committee for using recombinant DNA)
- Protocol number for the chimeric pups
- IT form for facility charges
General timeline
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ES cells delivered on day of injection session
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Eggs injected for 2-3 sessions
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Foster mice give birth (investigator notified)
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Chimeric pups are reported when coats come in
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Pups are weaned at 21 days and chimeric pups are released to the investigator
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The entire process normally takes 7-8 weeks
Special notes
The facility guarantees the production of 3 chimeric mice per cell line, however there is no guarantee that the chimeric pups are capable of germline transmission.
The facility routinely uses C57BL/6 mice from Harlan as the background strain. Investigators who need to use a different strain will separately purchase the male and female mice required for the procedure and pay the per diem charges for these animals.
If the investigator does not collect their chimeric pups by weaning (21 days), the pups will be transferred to the investigatorÕs protocol number and the investigator must pay the per diem charges for the pups.
The investigator must inform the facility if the ES cells are expected to produce severe defects or lethality, so that the pups can be properly monitored.
If your facility requires specific health tests before they will accept the transgenic pups, the investigator will pay for the cost of testing, including any shipping charges.
The current charge for ES cell injection is $1500 for Tulane investigators, $1800 for outside investigators.
Last update May 2008