CRISPR-mediated gene edited mice are created by injecting/electroporating CRISPR reagents into mouse zygotes. Transgenic founder mice are generated by injecting a transgene into mouse zygotes. Injected mouse zygotes will be implanted into foster females to carry them to term. Offspring will be transferred to investigator’s protocol for genotyping.
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A targeting vector will be introduced into ES cells via electroporation. ES cells will then be grown in appropriate selection media. Around 200 ES cell clones will be picked up, duplicated, and given to the investigator for analysis. Positive ES clones will then be expanded, frozen, and kept in the facility for subsequent blastocyst injection to generate chimeric mice.
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ES Cell Microinjection
Chimeric mice can be generated by microinjection of targeted ES cells into 3.5 days old mouse embryos. The facility will perform the microinjection and transfer injected blastocysts into foster families. Chimeric mice will be transferred to an investigator’s protocol after weaned. Since mouse ES cells are pluripotent and can contribute to all cells in an animal, including gametes, genetic alterations introduced into the ES cells can be propagated in the next generation to form a stable mutant mouse line.
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Mouse embryo or sperm can be cryopreserved to prevent accidental loss of a valuable mouse line or to discontinue the maintenance of a mouse colony. About 200 embryos per mouse line will be cryopreserved. Usually two males are required for sperm cryopreservation for each mouse line. In vitro quality control is included for both embryo and sperm freezing.
The Transgenic Mouse Facility will perform experiments to clean a mouse line from pathogen infections. The procedure will include superovulating, mating, and collecting fertilized one-cell embryos. The investigator is responsible the mouse purchase. Females are super-ovulated and bred to males of the line to be rederived. Embryos are collected and pooled, washed to further reduce the risk of pathogen transfer, and then surgically transferred, using aseptic technique, into the reproductive tract of pseudopregnant SPF females. Upon weaning of the pups, DAR will submit surrogate dams for diagnostic testing, including screens for bacterial and viral pathogens.
Mouse Line Recovery
A mouse line can be reconstituted from fresh or frozen embryos through the technique of embryo transfer. It also can be performed with fresh or frozen sperms through in vitro fertilization.
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The Transgenic Mouse Facility staff can train or assist individual mouse users in aspects of mouse colony breeding, such as weaning pups, extracting tail DNA, setting up timed mating, dissecting mouse embryos, and handling mouse ES cell cultures. These services are contingent upon the availability of time, equipment, animal health, and other factors.