Using this method, the creation of chimeric transgenic animals involves the microinjection of genetically modified pluripotent embryonic stem (ES) cells into the cavity of an expanded 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 genetically modified 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.
The client must supply at least one frozen vial of each ES cell clone to be injected. Each clone must have been tested for mycoplasma and mouse pathogens. We strongly encourage clients to have the average chromosome number determined for each clone. In our experience, any clone in which less than 50% of the cells are euploid is probably not worth injecting. Aneuploid cells can result in chimeras, but their genetic material rarely transmits through the germline. If this has not been done already, the TMII will require an extra vial of each clone for testing purposes.
We can expand a clone and freeze back multiple tubes of cells for an additional fee. If the client supplies a single vial for injection and does not ask us to expand it and freeze back extra vials, we will not be able to return any cells to the client after the injection. Clients have the option of providing freshly harvested cells on the day of injection.
The TMII can inject genetically modified ES cells for investigators who have either generated targeted cells in their lab or obtained genetically modified ES cell clones from another source. Investigator provided ES cells are injected into the cavity of an expanded blastocyst stage embryo and injected embryos are surgically transferred into the reproductive tract of surrogate pseudopregnant CD-1 recipient females. Once the chimeric pups are born, the TMII will nurture chimeric mice up to 21 postnatal days, at which point animals will be transferred to the investigator, who will be responsible for breeding and establishing the line of mice. Those that transmit the ‘knock-out’ allele to germline produce agouti pups, which are analyzed for phenotype by the client.
Chimeras are usually identified on the basis of coat color (but see below for exceptions). Some ES cell lines are derived from one of the various 129 mouse lines, which have agouti (brown) fur. Blastocyst donors, by contrast, are usually C57BL/6 mice, which have black coats. Thus, chimeras from these lines have a mixture of black and agouti fur, with the agouti patches arising from the ES cell lineage.
Not all ES cells microinjected into mouse embryos will produce high-degree chimeric founder animals nor contribute to the germline of the resulting mouse. The ability to generate germline transmitting chimeras rests solely with the pluripotent capacity of the microinjected ES cells. Keeping ES cells in a non-differentiated state is largely a function of how carefully the cells are treated/handled during culture.
For these reasons, when working with ES cell clones cultured outside our lab, we cannot guarantee that microinjections will result in the generation of chimeric animals that are transgenic.