The Animal Modeling Core meets with the investigator and/or a knowledgeable laboratory member to discuss the best strategy for generating the most appropriate animal model (species, genetic background, characterization, and timeline).
Design and generation of DNA targeting constructs
The AMC will design and assemble DNA targeting constructs to create the desired animal model. Production of targeting vectors to generate conditional knockout, knockin, and transgenic rats can be purchased separately or as part of a larger contract to generate the animal model.
Transgenic rat production via zygote injections
Donor rats are super-ovulated and mated to obtain one-cell stage zygotes. Transgenic rats are generated by pronuclear microinjection of engineered DNA into the zygotes. Manipulated embryos are then transferred into pseudopregnant surrogate dams.
Chimera rat production
Embryonic stem (ES) cells harboring a desired genetic manipulation are microinjected into blastocyst stage embryos. Manipulated embryos are then transferred into pseudopregnant surrogate dams. Chimeric rats can be generated using commercially available ES cells or by custom ES cell targeted via the AMC.
CRISPR/Cas9-mediated genome editing (KI/KO)
CRISPR/Cas9 technology can be used to produce gene knockouts (KOs) and some knockins (KIs) directly within the zygote. Guide RNA (gRNA), Cas9 mRNA/protein, along with repair DNA template (when appropriate) is microinjected or electroporated into one-cell stage zygotes. Manipulated embryos are then transferred into pseudopregnant surrogate dams.
Site-specific KI transgenic rats via integrase [COMING SOON]
Phi C31 integrases catalyze irreversible recombination between corresponding attB and attP sites. The AMC has engineered F344 rats with 2x attP landing sites knocked into the Rosa26 locus that are used as embryo donors. DNA constructs with corresponding attB sites are then microinjected into fertilized zygotes and incorporated into the Rosa26 locus in a single copy, controlled manner.