Embryonic Stem (ES) Cell-Based Gene Targeting
The Transgenic and Knockout Core can develop mice with targeted mutations in the entire mouse or in specific tissues or cell types. It normally takes about a year to generate a gene-targeted mouse.
Mice with targeted mutations are produced by first modifying a given endogenous gene locus in embryonic stem cells via homologous recombination. Embryonic stem cells carrying the desired gene mutation are then microinjected into blastocyst-stage embryos.
Microinjected embryos are transferred into pseudopregnant surrogate mothers that develop, deliver and nurse chimeric offspring. The chimeras are then bred to establish the knockout mouse line.
The core will generate gene-targeted embryonic stem cell clones, inject these clones into blastocysts and breed the resulting chimeras to obtain germline transmission of the targeted mutation.
Blastocyst microinjection service
Gene trapping in mouse embryonic stem cells is a powerful, cost-effective approach for creating large numbers of insertional mutations in mice. Transgenic and Knockout Core personnel can inject gene-trapped mouse embryonic stem cells into blastocysts, with the resulting chimeras then bred to germline transmission of the desired mutation.
This service operates on a first-come, first-served basis. Gene-trapped mouse embryonic stem cell lines are available from several sources.
Steps in the production of gene-targeted mice include:
- Construction of a gene-targeting vector from the isogenic genomic DNA
- Electroporation of the targeting vector into embryonic stem cells and isolation of drug-resistant embryonic stem cell clones
- Identification of gene-targeted embryonic stem cell clones
- Generation of embryonic stem cell mouse chimeras
- Germline transmission of the gene-targeted allele
Before being injected into C57BL/6 mouse blastocysts to generate chimeric mice, correctly targeted embryonic stem cell clones undergo karyotyping. Only clones with 40 chromosomes are injected. Ten metaphase spreads are analyzed per targeted clone. Three to six independently targeted embryonic stem cell clones are each injected into 16 to 40 blastocysts. Microinjections are normally completed within two to three weeks after the investigator submits a request.
If embryonic stem cells have been properly cultured, about half of the targeted embryonic stem cell clones will give rise to germline-competent male chimeras, with one to 10 chimeric males typically obtained per injected embryonic stem cell clone.
Chimeras are evaluated for the likelihood of germline transmission. At this point, investigators are advised to generate new gene-targeted embryonic stem cell clones if there is a low likelihood of germline transmission of the mutation.