Gene Targeting

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. Cells are then manipulated in culture and, when injected back into a blastocyst, the ES cells 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 cell and the ICM. The desired outcome of this process is to create chimeric mice that inherit germ cells from the injected ES cell genome. Using these techniques, 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.

TTML ES cell lines

The TTML routinely uses germline competent 129P2/Ola-Hsd derived HM1 ES cells (Magin, et. al. PDF; Selfridge, et. al. PDF) for gene targeting projects. These cells were originally created from the ICM of blastocysts isolated from E14TG2a mice and are HPRT deficient. When injected into blastocysts, they typically yield highly chimeric males at high frequencies and readily contribute to the germline of these animals.

Traditionally, 129-derived ES cell lines have been the most common lines used for gene targeting studies. Traditionally, these are robust lines, retaining pluripotent potential at high passages and, with relative consistency, generate germline competent chimeras at high frequencies. However, there is a growing knowledge of the molecular mechanisms that govern stem cell pluri-potentiality. New cell culture medias formulated to modulate these mechanisms have made it easier to develop ES cell lines from different genetic backgrounds, including C57BL/6. Although these newer B6 lines are not as typically 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.

Our lab has recently started working with the C57BL/6 ES cell line, PRX B6. Microinjection of non-targeted parental PRX B6 cells into B6(Cg)-Tyr^c-2J/J blastocysts, an albino C57BL/6 mouse strain, resulted in the generation of chimeric mice at a high rate (picture). These highly chimeric PRX B6 males were capable of transmitting ES cell-derived genes, often in the first generation. However, to date, we have limited germline contribution data for targeted clones.

ES cell targeting

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 microinjection into blastocysts to create chimeric animals

Parental ES cell lines are derived from the ICM of E3.5 embryonic stage blastocysts. Following in vitro manipulation, targeted ES cell clones carrying the desired mutation are then microinjected back into the same stage embryo from which they were derived, an E3.5 blastocyst. Once introduced into the blastocyst, pluripotent ES cells incorporate back into the ICM of the developing embryo. Coat color markers are used to determine the relative contribution of the ES cell genome to the genetic makeup of the chimeric animal (FAQ). Chimeric animals that transmit the (altered) ES cell genome, and thus the mutation, are used to study gene regulation and function, or mated to deletor mice to create conditional mutations in specific tissues.

Detailed Project Information

Phase I: ES Cell Targeting

Phase II: Production of Chimeric Mice

General information about targeting vector design

Site-specific DNA recombinases

Basic information about screening assay design (strategy)

References

1. Magin, T.M., McWhir, J., and D.W. Melton. A New Mouse Embryonic Stem Cell Line With Good Germ Line Contribution And Gene Targeting Frequency. (1992). Nucl Acids Res 20(14): 3795-3796. (PDF)
2. Selfridge, J., Pow, A.M., McWhir, J., Magin, T.M., and D.W. Melton. Gene Targeting Using a Mouse HPRT Minigene/HPRT-Deficient Embryonic Stem Cell System: Inactivation of the Mouse ERCC-1 Gene. (1992). Somatic Cell and Molecular Genetics 18(4): 325-336. (PDF)
3. Simpson E. M., Linder C. C., Sargent E. E., Davisson M. T., Mobraaten L. E., and Sharp J. J. (1997) Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nature Genet. 16, 19-27.
4. Threadgill D. W., Yee D., Matin A., Nadeau J. H., and Magnuson T. (1997) Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain. Mamm. Genome 8: 390-393.