Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.