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Mus musculus

ABSTRACT: Reduced diploidization and survival improvement of semi-cloned mice produced from androgenetic haploid embryonic stem cells

PROVIDER: PRJNA388933 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5641304_1.fastq.gz	Fastqsanger.gz
	SRR5641304_2.fastq.gz	Fastqsanger.gz
	SRR5641305.fastq.gz	Fastqsanger.gz
	SRR5641306_1.fastq.gz	Fastqsanger.gz
	SRR5641306_2.fastq.gz	Fastqsanger.gz

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Reduced diploidization and survival improvement of semi-cloned mice produced from androgenetic haploid embryonic stem cells

Project description:We found that the overall DNA methylation and hydroxymethylation in AG-haESCs are extremely low, and the downregulation of both de novo methyltransferase Dnmt3b and methylation maintenance enzyme Dnmt1was discovered responsible for this DNA hypomethylation. Further, our study discovered that the correction of DNA methylation can greatly reduce the incidence of diploidization and further improve the survival of semi-cloned mice produced from AG-haESCs.

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Project description:The androgenetic embryonic stem cells we generated carrying several genes mutations could be injected into the oocyte and produce the semi-cloned mice. Those semi-cloned mice could mimic several DM1-associated phenotypes. We try to identify if any off-target exist in those cells inducing by CRISPR/Cas9.

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Overexpression of Let-7a Mitigates Diploidization in Mouse Androgenetic Haploid Embryonic Stem Cells

Project description:Mouse androgenetic haploid embryonic stem cells (mAG-haESCs) can be utilized to uncover gene functions, especially those of genes with recessive effects, and to produce semicloned mice when injected into mature oocytes. However, mouse haploid cells undergo rapid diploidization during long-term culture in vitro and subsequently lose the advantages of haploidy and the factors that drive diploidization are not well understood. In this study, we compared the small RNAs (sRNAs) of mAG-haESCs, normal ESCs and mouse round spermatids by high-throughput sequencing and identified distinct sRNA profiles. Several let-7 family members and miR-290-295 cluster miRNAs were found significantly differentially transcribed. Knockdown and overexpression experiments showed that let-7a and let-7g suppress diploidization while miR-290a facilitates diploidization. Our study revealed the unique sRNA profile of mAG-haESCs and demonstrated that let-7a overexpression can mitigate diploidization in mAG-haESCs. These findings will help us to better understand mAG-haESCs and utilize them as a tool in the future.

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Project description:We reported a kind of new haploid embryonic stem cell, human haploid androgenetic embryonic stem cell, which kept the sperm characteristic epigenetic modification patterns for imprinting genes. In this study, two human haploid androgenetic embryonic stem cell lines (ha-AGHESC) and two human haploid parthenogenetic embryonic stem cell lines (ha-PGHESC) with somatic control and diploid HESC control, were processed with RNA-sequencing (RNA-seq) and whole genome bisulfite sequencing (WGBS). We showed that the reconstructed semi-clone HESCs were similar to the diploid HESC in transcriptome and the methylome especially related to the known human imprinting genes. The raw data of WGBS and bulk RNA-seq are deposited at Genome Sequence Archive (GSA) of Human with accession number HRA004100.

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Viable semi-cloned mice derived from androgenetic haploid stem cells lacking DNA methylation [bisulfite-Seq]

Project description:Viable semi-cloned mice derived from androgenetic haploid stem cells lacking DNA methylation [bisulfite-Seq]

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Project description:Androgenetic haploid embryonic stem cells

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Haploid embryonic stem cells

Project description:Mammalian haploid embryonic stem cells (haESCs) provide new possibilities for large-scale genetic screens because they bear only one copy of each chromosome. However, haESCs are prone to spontaneous diploidization through unknown mechanisms. Here, we report that a small molecule combination could restrain mouse haESCs from diploidization by impeding exit from naïve pluripotency and by shortening the S-G2/M phases. Combined with 2i and PD166285, our chemical cocktail could maintain haESCs in the haploid state for at least five weeks without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Taken together, we established an effective chemical approach for long-term maintenance of haESCs, and highlighted that proper cell cycle progression was critical for the maintenance of haploid state.

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