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CRISPR/Cas9 Genome Editing

The TMF now offers Cas9/gRNA mediated targeting as a service.  The service can include the design, engineering, QC testing, and microinjection (into fertilized mouse eggs) of a cocktail of the components (RNA and/or DNA) necessary to perform the desired editing at a locus pre-determined by the client.  Genome editing bypasses the traditional gene targeting steps involving ES cells and can even produce multiple mutations in the same founder animal. Please contact Jon Neumann for a consultation.


New Capabilities

Production of sex-sorted blastocysts

By mating wildtype females with male mice carrying an X-linked GFP transgene, we can produce blastocysts that can be sorted by sex (female embryos are fluorescent but male embryos are not).  When such blastocysts are injected with ES cells to make chimeric mice and the resulting pups are sexed at birth, we can immediately see if any sex conversion has taken place (e.g., by male ES cells being injected into female embryos, or vice versa), which is a strong indication of germline capability.

identification of female embryos using X-linked GFP transgene

Production of aggregation chimeras

We remove the zona pellucida from pre-blastocyst stage embryos and combine two embryos (or an embryo and a clump of ES cells) in a microwell to make aggregation chimeras (see photo).  Mutations that are lethal in the heterozygous state may be amenable to being studied in chimeras.

aggregation chimeric mice

Production of tetraploid embryos

The blastomeres in 2-cell embryos are fused with a pulse of electricity, producing a tetraploid embryo that will develop normally to the blastocyst stage.  If tetraploid blastocysts are injected with ES cells, the resulting pups will be virtually 100% ES cell-derived.  This can be used as an assay of the pluripotency of an ES cell line.

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