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CRISPR/Cas9 based targeted transgenesis at H11 locus

The Hipp11 (H11) locus is a "safe-harbor" locus close to the centromere of mouse chromosome 11. Transgenes integrated into this locus can be expressed at a more consistent level than at the similar ROSA26 safe-harbor locus on chromosome 6.  An additional advantage is increased efficiency of transgenesis at the H11 locus using CRISPR / Cas9 in conjunction with a plasmid based targeting construct of up to 10kb in size. Constructs can be easily inserted into the targeting vector using Gibson cloning. This technology has been developed for TMF users via a collaboration with Dr. Evgeny Kvon in the Dept. of Developmental & Cell Biology, School of Biological Sciences. Please contact the TMF for additional information.

Use of assisted reproduction to produce age-matched cohorts of genetically modified mice for experimental analysis

A common challenge with research using genetically modified mice involves production of sufficient numbers of age-matched experimental and control mice of the requisite genotype. Efficient breeding of mice requires extensive knowledge of mouse reproductive biology and behavior, including tips and tricks to optimize breeding efficiency and survival of newborn mice. Often, mice used in studies may not be closely age matched, which can introduce or increase experimental variability. A low number of biological replicates can result in insufficient power to identify significant effects or to evaluate sex as a biological variable. In many cases, these obstacles can be overcome through use of assisted reproduction. Here, superovulation, in vitro fertilization and transfer of pre-implantation embryos to the reproductive tract of pseudo-pregnant donor females is used to produce relatively large cohorts of age matched animals of experimental and control genotypes for experimental analysis. Several investigators at UCI have benefitted from use of assisted reproduction to generate cohorts of animals for analysis. Please contact the TMF if you are interested in learning more about this strategy and whether it might help accelerate your research, reduce the cost of doing so and reducing the overall numbers of animals required.

Improved Services

Hyper-ovulation of egg donors

To continue to address the 3R's and to reduce the cost of our services, the TMF uses hyper-ovulation to produce oocytes and zygotes for different services. The difference between conventional superovulation and hyperovulation is the inclusion of an antibody against inhibin, which is administered along with the PMSG. During superovulation, exposure to FSH induces synthesis of inhibin which inhibits secretion of endogenous FSH from the anterior pituitary. Addition of antisera against inhibin blocks this process, producing an increase in oocytes recovered per mouse. For example, during re-derivation via IVF, the TMF can reduce the number of females used as egg donors from 12-14 down to 5 - 6, a 50% reduction. We obtain our hyperovulation reagents from Cosmo Bio Ltd. See Takeo & Nakagata (2015) PLoS One 10: e0128330 for more details. 

Innovative Strategies

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

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