Generation of Genetically Modified Mice by Oocyte Injection of Androgenetic Haploid Embryonic Stem Cells

Abstract

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

“…In the mouse study, approximately 16-21% of haploid zygotes developed to the blastocyst stage, which was significantly lower than was observed for the diploid zygotes. 7 This trend is in accordance with our results.…”

“…7 Therefore, haploid androgeneic ES cells provide a potential method to resolve infertility caused by azoospermia and could also be used as a transgenic tool. Several labs have reported haploid mouse ES cells, [5][6][7] but not haploid human ES cells, thereby limiting further application in humans. One key limitation lies in obtaining haploid embryos or blastocysts for ES derivation.…”

“…In mice, two approaches have been used to derive haploid embryos, including injecting one sperm while removing the oocyte's chromosomes or removing the female pronucleus from the zygote. 7 However, in humans, both approaches require MII oocytes, which are difficult to obtain due to ethical issues.…”

Generation of human haploid embryonic stem cells from parthenogenetic embryos obtained by microsurgical removal of male pronucleus

“…Haploid embryonic stem cells (haESCs) have been recently generated from parthenogenetic (PG) or androgenetic (AG) blastocysts of different mammals, including mouse, rat and monkey, enabling genetic screening at both cellular and organism levels [1][2][3][4][5][6]. However, whether haESCs can be generated from human remains unknown.…”

Gametogenesis in a dish

“…Sperm derived from these transplanted germ cells are capable of fertilizing oocytes in vitro, and further generating viable offspring that are developmentally normal and fertile [10]. More interestingly, a very recent study showed that androgenetic haploid mouse ESCs can be derived and genetically manipulated in vitro [11]. Interestingly, these cells, when injected into oocytes, can give rise to fertile adult mice, suggesting that these sperm-like ESCs can be used as a genetically tractable alternative for in vitro fertilization [11].…”