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ES cell selection and maintanence | Procedural Outline

The Gene-Targeted Mouse Service was established by Tom Doetschman, Ph.D. at the University of Cincinnati to develop genetically altered mice at a cost below that required for individual preparation. The Service Core is available to investigators within and outside the University of Cincinnati. The use of the Service by investigators outside the University is encouraged. At present, approximately 50% of our work is from the outside.

The Service Core facilities:

• Office/Laboratory:

CO2 incubator, containment hood, flouroscope, computerized database.

• Microinjection room:

2 Laminar flow hoods and 2 Zeiss stereoscopes for preparation of mouse blastocysts. Two Nikon Diaphot and one Zeiss microscope with Narashigi micromanipulators for injection of ES cells. Needle puller and microforge for needle preparation.

• Implant room in pathogen-free barrier facility:

2 laminar flow hoods. 2 Zeiss stereoscopes. Sterile surgical instruments.

• Mouse housing suite in a pathogen-free barrier facility:

Housing for 3500 mice in microisolator cages, with up 1000 cages devoted to the Service Core. Racks for carrying sterile water and air. Three laminar flow hoods for cage/mouse manipulation.

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• Health reports:

The Service uses sentinel mice in the barrier facility. These sentinel mice are assayed on a regular basis. Health reports are also obtained on those mice purchased by the Service on a regular basis: C3HBl/6 orNIH Swiss black foster mothers, C57Bl/6 males and females for superovulation, NIH Swiss black females used as mates. Any change in the health status of the mice in the barrier facility is reported to those investigators using the Service.

• Technology:

Four weeks of the year are set aside for tests in improving the efficiency of production of chimeric, germline mice. These efforts focus on assays to select those ES cells having the potential for germline mouse production and on technical improvements to increase mouse production. Such improvements are passed onto the investigator in the form of lowered costs.

• ES cell selection and maintanence:

The Service can advise the investigators on the choice of ES cells used for gene-targeting based on our own experience. We try to keep current on the information dealing with the various strains of ES cells used. We can give advice on maintaining ES cells in the un-differentiated state.

• Access to the Service by investigators:

At this time, use of the Service is on a first come basis. Over the past year no investigator either inside or outside the University has had to wait more than six weeks from the time of submission of frozen clones to the initiation of injection. We approach scheduling on the basis of communication with each investigator. Dates for use of the Service set too far in advance are discouraged. The biology and technology involved in the investigators progress make planning too far in advance uncertain.

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• Procedural Outline:

1. Microinjection of targeted ES cells will be done between one to six weeks after the receipt of the ES cells.

2. Prior to injection, the ES cells must be tested for the absence of mycoplasm contamination. If supplied with 5-10mls of medium from ES cells grown under the required conditions, representative clone of ES cells grown for 3 weeks in the abscence of antibodies, the Service will assay for contamination. Any medium sent to us should not be frozen. Testing of media samples for mycoplasm generally takes 1-2 weeks, when done by the Service Core. After which time the investigator will be notified of the results by either fax or phone.

3. One to four clones of targeted ES cells can be sent to our facility in frozen vials on dry-ice by express mail. The shipping address for both mycoplam samples and ES cells is located on the information form, which should accompany any materials sent to the Service. Additional information on shipping can be found on the shipping page of this site.

4. The gene-targeted ES cells will be injected into blastocysts obtained post fertilization of C57Bl/6 female mice; injected blastocysts will be transferred to the uterus of pseudopregnant females. Coat color chimeric progeny mice will be bred to black coat mice to determine if the 129 strain, ES cell genotype, is passed through the germline: germline chimeras are defined as producing progeny with an agouti coat.

5. Tail clips from the agouti F1 offspring are sent to the investigator as soon as possible. Conditions for the transfer of mice to the investigator is arranged by phone.

John Duffy, Ph.D., Director.

Tom Doetschman, Ph.D., Senior Advisor/Consultant

 

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University of Cincinnati

Gene-Targeted Mouse Service

Phone: (513)558-5314

Fax:(513)558-3181