Pluripotent embryonic stem (ES) cells were developed as a tool for introducing specific, site-directed alterations into the mammalian genome to create mouse models in which gene function and regulation can be studied. ES cell lines are derived in vitro from the outgrowth of the inner cell mass (ICM) of a blastocyst, the portion of the blastocyst that gives rise to the embryo. Undifferentiated ES cells are then manipulated in culture and, when injected into a blastocyst, have the ability to incorporate back into the ICM and contribute to the genetic makeup of the developing embryo. The resulting pups are considered chimeric in their genetic makeup as they consist of tissues deriving from both the ES cells and cells within the ICM of the blastocyst. The desired outcome is to create chimeric mice that inherit germ cells derived from the injected ES cells. In this way, mutations can be introduced into the ES cells in vitro, then incorporated in vivo into the germline of a mouse and transmitted from generation to generation.
Our services include:
- ES Cell Targeting
- Production of Chimeric Mice
- Targeting Vector Design
- Screening Assay Design
- Mating TTML Animals
Learn about our services and next steps via the links and information below:
- Gene Targeting Project Initiation
Learn how to begin a project and work with TTML staff.
- Services & Rates
Get the run-down on fees and the services they cover.
About ES Cell Lines
Traditionally, 129-derived ES cell lines have been the most common lines used globally for gene targeting studies. In general, 129-derived ES cell lines are robust lines, retaining pluripotent potential at high passages and, with relative consistency, generate germline competent chimeras at high frequencies. The R1 (129X1 X 129S1) and E14 (129P2/OlaHsd) lines are two of the most popular and well characterized 129-derived ES cell lines.
The rapidly growing understanding of the molecular mechanisms that govern stem cell pluri-potentiality and the newer cell culture medias formulated to modulate these mechanisms have made it easier to develop ES cell lines from different genetic backgrounds, including C57BL/6N (B6N). Although B6N lines are usually not as robust as 129 lines, the C57BL/6 genome has been completely sequenced and there are a greater number of isogenic resources such as C57BL/6 BAC libraries available for targeting vector design. Homologous recombination in E. Coli (recombineering), which requires C57BL/6 BAC DNA, can be used to rapidly construct targeting vectors.
In addition, some 50,000 targeted alleles have been generate through the KOMP1 initiative, all in the B6N-derived JM8 ES cell line.
Creating Targeted Alleles in ES Cells via Homologous Recombination
Mutations are introduced into ES cells following electroporation of a DNA targeting vector. Pairing of the homologous region targeting vector sequences with the endogenous gene leads to site-specific integration of targeting vector DNA into the genome via homologous recombination and the concurrent loss of the endogenous gene sequences. A targeted event is highly dependent on the locus/gene being targeted and occurs infrequently for most projects. Unless previously established, there is no way to predict the susceptibility of a gene or locus to targeting, and, therefore, hundreds of ES cell clones must be selected and genotyped.
ES Cell Lines Available for Our Gene Targeting Projects
Three ES cell lines are routinely available for our gene targeting projects: PRX-B6 (C57BL/6N), PRX-129 (129S6/SvEv) and G4 (129S6 X B6N F1 hybrid, Nagy Lab). When injected into blastocysts, our standard ES cell lines typically yield highly chimeric males at high frequencies and readily contribute to the germline of these animals. Highly chimeric males are generally capable of transmitting ES cell-derived genes, often in the first generation. See our track record page for more details.
1. Simpson E. M., et. al (1997) Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nature Genet. 16, 19-27.
2. Threadgill D. W., et.al (1997) Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome 8: 390-393.