Microinjection Services

The Microinjection (MIJ) Service is comprised of the facilities, instrumentation, and technical expertise necessary to carry out microinjection, an essential step in the process to create a genetically modified mouse model. The MIJ staff carries out the daily activity of mouse colony management, superovulation, embryo harvest, pronuclear, cytoplasmic, and blastocyst injection and embryo transfer surgery. Service offerings include microinjection of CRISPR reagents or a DNA construct, including BAC (bacterial artificial chromosome), into the zygote of many inbred and specialty strains of mice. We also offer ES cell injection into both blastocyst and 8-cell stage embryos of conventional mouse strains and coat color scheme is used whenever possible to facilitate assessment of chimerism. The MIJ Services has also been very successful in creating knockout animal models using ZFN (Zinc Finger Nuclease) and TALEN (Transcription Activator Like Effector Nuclease) technologies directly on inbred and specialty strains. The Microinjection Service currently has 5 complete microinjection stations and 3 complete surgical stations. Key equipment includes: 3 VetEquip anesthesia machines, 3 XYClone laser systems, 14 stereomicroscopes, 6 benchtop incubators, 2 Sutter and 2 Kopf micropipette pullers, 2 Piezo drills and 3 microforges.

Mouse Model Production:

• Knockout as well as knockin mouse models are produced by injecting CRISPR reagents into the zygotes (0.5 dpc) of the desired host strain. These embryos develop to term after surgical transfer into a recipient CByB6F1/J pseudopregnant female mouse. The guide RNA recognizes the genomic locus by base pairing, and the Cas9 nuclease generates a double strand break (DSB) in the genome. If no donor DNA is used, the DSB is repaired by the error prone mechanism of nonhomologous end joining (NHEJ) which often carries a frameshift mutation. However, when donor DNA is provided and used, homology directed repair (HDR) will mediate incorporation of a point mutation, insertion of a tag or a loxP site or swapping of the murine gene with its human ortholog.

• Transgenic mice are produced by microinjecting a purified gene construct into the pronucleus of zygotes (0.5 dpc) of the desired host strain. These embryos develop to term after surgical transfer into a recipient CByB6F1/J pseudopregnant female mouse. The resulting pups are transferred to the investigator for identification of transgene-carriers. These "founder" mice are then bred by the investigator for expression analysis.

• Targeted-mutant mice are produced by microinjecting genetically-modified embryonic stem (ES) cells into blastocyst-stage mouse embryos (3.5 dpc). After surgical transfer and development to term, chimeras (derived from both the host embryo and the introduced cells) are identified by coat color and bred by the investigator for germ-line transmission of the manipulated ES cell lineage.