Production of Chimeric Mice

Creation of germline competent chimeric animals involves the microinjection of pluripotent embryonic stem (ES) cells into the cavity of an expanded 3.5 d.p.c. blastocyst stage embryo. ES cell lines are initially derived in vitro from the outgrowth of the inner cell mass (ICM) of the blastocyst, the portion of the blastocyst that gives rise to the embryo. Thus, when injected back into a blastocyst, the ES cells have the ability to incorporate 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 microinjected ES cell and the endogenous ICM of the host blastocyst genome. The desired outcome of this process is to create chimeric mice that inherit germ cells derived from the microinjected ES cells. Using this technique, 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.

There is no way to predict a priori the pluripotent potential of a given ES clone prior to microinjection. While clones that look beautiful in culture exhibiting characteristics of undifferentiated cells is promising, visual appearance alone is not indicative of pluripotency. Individual ES cell clones vary in their ability to contribute to the genetic makeup of a chimeric mouse. Good clones are capable of consistently generating highly chimeric males while other clones may generate only low percentage chimeras or none at all.

A chimera is defined by Wikipedia as an animal that has two or more different populations of genetically distinct cells that originated in different zygotes; if the different cells emerged from the same zygote, it is called a mosaic (which happens occasionally in transgenic founders but not in ES cell derived chimeras).

ES cell chimeras are comprised of cell populations and tissues arising from both the host blastocyst strain and the ES cell clone introduced into the E3.5 blastocyst stage embryo. While ES cell contribution to organs and tissues is not immediately evident and must be determined experimentally, ES cell contribution to pigmented tissues such as eyes, skin and hair can be visualized in neonatal pups. An indication of ES cell contribution to coat color can be visualized at about postnatal day 10 when the hair begins to come in. For HM1 (129P2/Ola) derived pups, ES cell contribution is notable as early as postnatal day 3 as marbled color skin patterns emerge. For some C57BL/6 ES cell derived pups, chimerism is visual at birth by eye color.

The percent chimerism of each mouse is determined at weaning and refers to the degree of coat color deriving from the ES genome. The greater the ES cell contribution, the higher the percent chimerism. For example, a 95 percent 129-derived chimeric male will have only about 5 percent pure black color inherited from the host blastocyst cells and the rest will be non-black.

Coat color chimerism indicates that individual ES cells were incorporated into the ICM of the blastocyst following microinjection and that, once incorporated, were capable of giving rise to various cells or tissues in the resulting animal.

As both germ cells and melanocytes (pigment producing cells of the skin, hair and eyes) arise from migratory precursor cell populations within the developing embryo, the percent of 129 ES cell derived coloration in the coat is often correlative with the inheritance of ES cell-derived germ cells. For example, 129P2/Ola derived chimeras have marbled coats (and skin) of chinchilla, white and agouti (Mouse Strain Coat Colors). Thus, the higher percentage of ES cell derived coat coloration, or chimerism, the greater the likelihood that the incorporated ES cells were also able to generate germ cells that migrated to and populated the germinal ridges.

Coat color chimerism and germline transmission is less correlative with C57BL/6 ES cell lines.

No. Each chimeric mouse, even those created from the same ES cell clone and regardless of percent chimerism, is unique in its ability to transmit ES cell inherited genes. Germline transmission of ES cell derived genes in 129 derived chimeras mated to wild-type C57BL/6 is indicated by the production of agouti pups. It is not unusual for highly chimeric males (90-99 percent chimerism) to be sterile, or to produce many black pups before generating agouti pups. Some fertile males will never produce any agouti pups. In our experience, highly chimeric HM1-derived males often, but not always, generate agouti pups in the first or second generation. Agouti coat color only indicates the ability of the ES cell to give rise to germ cells and agouti pups must be genotyped for the inheritance of the mutated allele.

Chimeric mice can be mated with virtually any strain desired. However, we recommend mating highly chimeric males to wild-type females of the blastocyst strain initially to assess the frequency with which ES cell derived genes are transmitted by a particular chimera. This is particularly true for conditional KO lines. Coat or eye color of offspring indicate germline transmission when blastocyst strain animals are mated with 129-derived and B6-derived chimeras, respectively. When using these strain combinations, only the pups with ES cell derived coloring need to be genotyped.

See mating schemes for 129- and B6-derived chimeras.

F1 mice carrying the mutated allele or chimeric mice known to transmit the mutation at high frequency should be used for further mating or to create conditional KO animals.

If using other mating schemes or strains in which color cannot be used as a indicator of ES cell germline transmission, all pups must be genotyped.

No. Much simpler and faster PCR-based genotyping strategies should be used at this point. The elaborate screening assays developed to detect 5' and 3' targeting events in the ES cell clones are not necessary at this stage, as homologous recombination in a particular ES cell clone should have been confirmed prior to microinjection.

When starting, keep in mind that each chimeric male, even those created from the same ES cell clone, is unique in its ability to transmit ES cell inherited genes. It is not unusual for chimeric males to be sterile or to produce many black pups before generating agouti pups. Some fertile males will never produce any agouti pups. Therefore, it is advantageous to begin breeding several of your best males initially. It is also wise to breed males generated from at least two different clones if available. View general mouse mating schemes.

In general, highly chimeric females should be bred only if male chimeras are not available. Females occasionally produce agouti pups, but the frequency is usually very low. However, if females are the only chimeras available, by all means, give it a go.