Center for Genetic Medicine

The Transgenic and Targeted Mutagenesis Laboratory is designed to produce genetically modified mice for research projects of faculty on the Evanston campus, at the Medical School and at Children’s Memorial Research Center (CMRC). This shared resource serves as a centralized infrastructure possessing both the necessary equipment and technical expertise for the production of transgenic animals by pronuclear injection and targeted mutagenesis of embryonic stem (ES) cells. Transgenic animals are produced by pronuclear microinjection of purified transgenic DNA. Injected embryos are surgically transferred in the oviduct of surrogate mothers and the resulting offspring are screened for the inheritance of the transgene. Gene targeting services, via homologous recombination in mouse ES cells, are available to investigators interested in generating genetically modified mice carrying targeted mutations that disrupt gene activity (knock-out mice) or introduce site-specific mutations into the genome (knock-in mice). The HM1 (129/Ola) ES cell line is the principle line maintained by the Facility although additional lines have recently been acquired. The Facility can provide primary construction vectors to interested investigators and advice on vector design and screening strategies. Currently, however, the client lab is responsible for generation and purification of targeting vectors. Following electroporation of the targeting vector and drug selection, DNA from 250-480 colonies is provided to the investigator for integration analysis. Identified clones are expanded and confirmed for correct targeting. Chimeric mice are generated from targeted ES clones, generated either by the Facility or provided by an investigator, injected into the blastocoel cavity of E3.5 blastocysts that are then surgically transferred into the reproductive tract of pseudopregnant females. Among the resulting litters are colorful chimeric pups with tissues, including germ tissue, composed of cells derived from both the targeted ES cells (white/agouti in color) and the host blastocyst (black in color). Chimeric mice are given to the client lab for breeding to identify ES cell derived offspring that are heterozygous for the targeted mutation.