Abstract
In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.
Highlights
The genetic information of an oocyte and a spermatozoon is passed onto the offspring
In order to manipulate genomic imprinting in parthenogenetic haESCs (phaESCs), the CRISPR-Cas9 system was used to delete the IG-differentially methylated regions (DMRs) and H19-DMR in a phaESC line that was established from a 129S6/ SvEvTac mouse oocyte (Fig 1A and S1A and S1B Fig)
After transfection with expression vectors for CRISPR-Cas9 nucleases and guide RNAs, a piggyBac transposon plasmid for EGFP expression, and an expression vector for a piggyBac transposase, single cells were plated into multi-well dishes to establish clonal cultures. 2 double-knockout phaESC lines, DKO-phaESC1 and DKO-phaESC-2, were identified by PCR-based genotyping (S1C Fig)
Summary
The genetic information of an oocyte and a spermatozoon is passed onto the offspring. Both maternal and paternal genomes are required for normal development of mammalian embryos. Uniparental embryos suffer developmental defects due to the imbalance of genomic imprinting [1]. Despite the importance of the gametic genome, the prerequisites for forming a totipotent zygote remain incompletely understood.
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