Abstract
The Black and Tan Brachyury (BTBR) mouse strain is a valuable model for the study of long-term complications from obesity-induced type 2 diabetes mellitus and autism spectrum disorder. Due to technical difficulties with assisted reproduction, genetically modified animals on this background have previously been generated through extensive backcrossing, which is expensive and time-consuming. We successfully generated two separate transgenic mouse lines after direct zygote microinjection into this background strain. Additionally, we developed in vitro fertilization (IVF) methods for the BTBR mouse. We found low rates of fertilization and implantation in this strain, and identified the BTBR oocyte as the primary culprit of low success with BTBR IVF. We achieved an increase in live born pups from 5.9 to 35.6 % with IVF in the BTBR strain by use of BTBR females at a younger age (18–25 days), collection of oocytes 15–17 h after superovulation, and the use of supplemented fertilization media. This method eliminates the need for time consuming assisted embryo manipulations that are otherwise required for success with BTBR oocytes. This advancement provides an exciting opportunity to directly generate BTBR transgenics and gene-edited mice using both traditional and emerging genomic editing techniques, such as CRISPR/Cas9. These methods also allow effective colony preservation and rederivation with these strains. To our knowledge, this is the first report describing embryo manipulations in BTBR mice.
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Acknowledgments
We thank Alan Attie, William Dove and Alexandra Shedlovsky for initial insights into work with the BTBR strain.
Funding
This work was supported by the National Institutes of Health training grants T32 RR023916 and T32 OD010423 (MB). DAF was supported by National Institute of Aging Training Grant T32 AG000213 and by the University of Wisconsin-Madison Science and Medicine Graduate Research Scholars Program. DBD is supported by NIDDK DK083442 and the University of Wisconsin Department of Medicine. This work was performed with resources from the William S. Middleton Memorial Veterans Hospital. This work does not represent the views of the Department of Veterans Affairs or the United States Government.
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The authors declare that they have no conflict of interest.
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.
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Baan, M., Krentz, K.J., Fontaine, D.A. et al. Successful in vitro fertilization and generation of transgenics in Black and Tan Brachyury (BTBR) mice. Transgenic Res 25, 847–854 (2016). https://doi.org/10.1007/s11248-016-9974-0
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DOI: https://doi.org/10.1007/s11248-016-9974-0