Androgenetic haploid embryonic stem cells produce live transgenic mice

Abstract

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.

Birth of fertile bimaternal offspring following intracytoplasmic injection of parthenogenetic haploid embryonic stem cells

“…Generation of gynogenetic bimaternal mice containing two sets of maternal genomes was achieved using non-growing oocytes with imprinting modifications [4]; however, the approach was technically challenging and impractical for further applications. Recently, we and others have derived mammalian androgenetic and parthenogenetic haploid embryonic stem cells (ahESCs and phESCs), and showed that ahESCs can replace gametes to produce offspring [5][6][7], which provided alternative resources for reproduction [6,7]. Here we report that after proper imprinting modifications, the mouse phESCs can efficiently produce viable fertile offspring upon intracytoplasmic injection into MII oocytes.…”

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

“…Spermatozoa were released from the cauda epididymis with forceps compressing [50] and suspended in M2 medium. ICSI was then carried out as described previously [51][52][53] with some modifications. Briefly, 10 µl of sperm suspension was added to 1 ml M2 medium supplemented with 5 µg/ml of cytochalasin B, and 15-20 mature oocytes were placed in the M2 medium for 5 min.…”

Generation of fertile offspring from Kitw/Kitwv mice through differentiation of gene corrected nuclear transfer embryonic stem cells

“…ICSI was performed as described previously [37]. Briefly, the haploid round sperm cell suspension was added into 1 ml M2 medium supplemented with 5 µg/ml of cytochalasin B.…”