KOMP/EUCOMM Blastocyst Injection
The ability of mouse pre-implantation embryos to incorporate cells from other embryos is fundamental to creating mutant mice. Thus when embryonic stem (ES) cells that have been genetically altered are microinjected into mouse E3.5 day embryos, mutant chimeras can be obtained. Since ES cells are pluripotent and can contribute to all cell types of an animal, including gametes, genetic alterations introduced into the ES cell genome can be propogated in the next generation to form a stable mutant mouse line. KOMP and EUCOMM cells are generated on an inbred background strain that requires special media and handling.
Genetically modified ES cells will be injected into C57Bl/6 or C57Bl/6 albino blastocysts. Injected blasts will then be transferred back to the uteri of foster females. Approximately three weeks after pups are born, facility personnel will inform investigators of the number of chimeras produced and their percentage chimerism. Chimeras selected by the investigator will then be transferred to the PI's animal protocol and animal colony. We have found that it can take several sessions to obtain chimeras from KOMP/EUCOMM cells. Thus, we will inject up to 100 blastocysts for each line.
$6000/cell line for MD Anderson investigators; $16,000/cell line for external investigators
Time Line for Blastocyst Injection
- Day 1: Microinject ES cell clone
- Day 17: Pups born
- Day 31: Investigator notified about births, male/female numbers, number of chimeras and % of chimerism. Investigator is responsible for turning in an animal order form for these mice to Veterinary Medicine at this time
- Day 38: Wean chimeras and transfer to investigator (upon receipt of signed order from Veterinary Medicine)
To Obtain Service from the GEMF
- Obtain authorization for animal research from the Office of Research Administration (animal protocol coordinator at Ext. 2-3220). The facility cannot initiate any experiment without an authorized animal research protocol number
- Schedule an appointment with Jan Parker-Thornburg (713-745-2654 or e-mail firstname.lastname@example.org) on North Campus, or Chen Dong (713-563-3263 or e-mail email@example.com) on South Campus, to set an injection date and for briefing on how to prepare ES cells for injection
- Complete and submit a service request form for blastocyst injection along with a Southern blot of the ES cell clones to be injected. Also complete and submit the Contract for Services for Cells Produced Outside of the GEMF, included on the service request form
Service request form for MD Anderson investigators (pdf)
Service request form for external investigators (pdf)
- Provide the facility with ES cells prepared according to the specified protocol on the day of injection
- Obtain and complete an authorization form with Veterinary Medicine for animal orders
Chimeras will be transferred to the investigator upon weaning for subsequent breeding.
Read the Policies and Procedures section for important information regarding services rendered by the GEMF.
Note: The likelihood of a targeted ES cell clone contributing to the germ-line is highly dependent on the quality of the ES cells injected into the blastocyst. It is therefore essential that ES cells for injection are maintained under optimal conditions and in an undifferentiated state throughout their culture. We maintain the right to refuse injection of cells that are differentiated, or which show aberrant characteristics.