Fertilizable Rat Sperm Is Generated in Mice Using Blastocyst Complementation: An Efficient Method for Producing Rats With ES Cell Traits

Abstract

We developed a novel approach for generating rat offspring using rat embryonic stem (ES) cell-derived sperm produced in mice with the blastocyst complementation method. By optimizing culture conditions, we established naïve male rat ES cells from two transgenic rat strains expressing EGFP and Venus fluorescence, respectively. The pluripotency of these cells was confirmed by the formation of germline chimeras. These ES cells were then injected into blastocysts of germ cell-deficient mice, which resulted in chimeric mice with the ability to produce rat-derived sperm. Histological analysis confirmed the presence of seminiferous tubules and spermatozoa, which are morphologically characteristic of rats, in the chimeric testes. To evaluate the fertilization potential of the chimeric mouse sperm, we performed intracytoplasmic sperm injection (ICSI) to rat oocytes and successfully produced viable offspring carrying ES cell-derived traits. This method eliminates concerns regarding host cell contribution, as all sperm in the chimeras originate from rats, enabling the use of nonfluorescent cells. Furthermore, the absence of competition with host cells is expected to enhance sperm production efficiency. By utilizing germ cell-deficient mice as recipients, this approach offers a cost-effective and efficient strategy for generating genetically modified rats, addressing key limitations in rat ES cell-based genetic engineering.