Reproductive Physiology as a Foundation for Developing Transgenic Peromyscus

Abstract

Peromyscus mice evolved to thrive in a wide range of habitats across North America, resulting in a high degree of diversity within and between species. Much work has already been done to understand the genetic bases of this variation, and advances in genetic engineering have enhanced our ability to target genes of interest. However, implementing CRISPR technology in a new species requires addressing hurdles in reproductive biology. Here we developed reproductive tools in P. maniculatus (deer mice) that will serve as the foundation for genome editing in this species. First, we characterized the estrous cycle length, composition, and endocrinology in deer mice. We found that P. maniculatus have a longer estrous cycle than laboratory mice (Mus musculus) as well as gonadotropin concentrations that are higher than in Mus and comparable to the closely related hamster (Mesocricetus auratus). Second, using a multifactorial design, we tested superovulation parameters to maximize oocyte yield in deer mice. We found that the ideal interval between gonadotropin doses is 56 hours, as is seen in hamsters and voles (Microtus), rather than the 48-hour interval that is typically performed in Mus. We also determined that hCG dose is the most important of the tested parameters. Based on the high baseline LH levels in deer mice and their similarity to those seen in hamsters, we recommend further testing gonadotropin doses at the high levels used in hamster superovulation protocols. Together, these two experiments provide the foundation that will enable us to test adaptive hypotheses in Peromyscus and, more generally, provide a framework for developing transgenics in other small mammal species with unique reproductive physiology.