The production of chimeric mice by introducing genetically modified Embryonic Stem (ES) cells into mouse blastocysts (3.5-day embryos) is an alternative approach to generating genetically engineered mice.
What the Facility will Provide
Through this service, the facility will generate chimeric mice by injecting Embryonic Stem (ES) cells containing site-specific genomic alterations into normal blastocysts. ES cells will be injected into a minimum of 50 blastocysts of C57BL/6 or Balb/c. When the blastocysts have re-expanded, they will be transferred into the uterine horn of pseudopregnant female mice and allowed to reach term. Upon weaning ~3 weeks after birth, chimeric mice are separated and then released to the care of the investigator.
Since this approach is time-consuming, the following guidelines are recommended:
- Prior to initiating the project, the potential of the parental (non-recombinant) ES cell line to generate high quality chimeras with germ line contribution should be confirmed.
- A mycoplasma test of the recombinant ES cell line must be performed since the presence of such contamination has been associated with fetal loss and reduced litter size.
- On the assigned day, the freshly harvested ES cells (free of feeder cells) will be delivered to the Core for injection.
- Documentation of the recombinant ES cell line screening results (Southern blot or PCR) should be submitted along with the chimeric mouse request form.
- A karyotype analysis of the recombinant clone is recommended. High levels of aneuploidy markedly decrease the success rate of this procedure.
Investigator’s Responsibilities
The investigator is responsible for maintaining the recombinant ES clone under conditions that inhibit their differentiation. Once the chimeric animals are weaned, it is the responsibility of the investigator to breed them for germ line transmission. It is recommended that you:
- Contact Kathleen Moosbrugger (kthompso@pennmedicine.upenn.edu – 215-573-3023) to arrange for testing the non-recombinant ES cell line and discuss the schedule of injection.
- Complete Online a service request form and submit evidence of approval of the IACUC and IBC committees for the project.
- Upon notification of a prearranged schedule, provide the facility with a freshly harvested sample of ES cells along with an aliquot of HEPES-buffered ES cell culture medium (5 ml) to be used for the injection chamber. The cell sample should be free of feeder cells. Additional information on maintaining and harvesting ES cells could be found in Teratocarcinomas and Embryonic Stem Cells (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, as well as in Meth. Enz. v. 225, 1993.
- Undertake the care of the weaned 3-week-old chimeric mice submitted to the project scientist. These mice can no longer reenter the barrier colony. It is the responsibility of the principal investigator to arrange for animal housing.
- Relay to the facility any information about germ line transmission once such information becomes available.
