ES Cell Injection
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 transgenic 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 and allowed to go to term. Upon weaning 3 weeks after birth, chimeric mice are separated and then released to the care of the investigator.
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 injection of the recombinant cell line, normal non-recombinant ES cells that are being used in the study should be tested for their chimera-producing potential.
- A mycoplasma test of the recombinant line is absolutely necessary since the presence of such contamination has been associated with fetal loss and reduced litter size.
- On the assigned day, the freshly harvested ES cell sample (free of feeder cells) will be delivered to the Core to proceed with this injection.
- A photocopy of the recombinant ES cell line screening results (Southern blot or PCR) should be submitted along with the chimeric mice 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 testing. It is recommended that you:
- Contact Jean Richa (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 at: http://www.med.upenn.edu/apps/my/transcore/user and submit evidence of approval of the IACUC and IBC committees for the project. Unless you have been involved with chimeric mouse work, this will require a new submission to both committees.
- 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.
Please also consult additional guidelines related to this service under the “Protocols” section of this site.