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The dog is recognized as a highly predictive model for preclinical research. Its size, life span, physiology, and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model has not been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for preclinical models of genetic diseases. Specifically, we i) established markers for identification and tracking canine spermatogonial cells; ii) established methods for enrichment and genetic manipulation of these cells; iii) described their behavior in culture; and iv) demonstrated engraftment of genetically manipulated SSC and production of transgenic sperm. These findings help to set the stage for generation of transgenic canine models via SSC transplantation.

Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line