Project description:This SuperSeries is composed of the following subset Series: GSE35785: mRNA expression data from AG-haESC, E14 and MEF GSE35786: CGH analysis of AG-haESCs (androgenetic haploid embryonic stem cells) Refer to individual Series
Project description:Haploid cells are amenable for genetic analysis because they contain only one set of chromosomes.Here,we report the derivation of haESCs from androgenetic blastocysts. These cells, which we designated AG-haESCs, express classical ESC markers, are pluripotent, and contribute to various tissues including the germline upon injection into diploid blastocysts. We used microarrays to compare the gene expression levels among androgenetic haploid embryonic stem cell lines(AG-haESC) E14 and male mouse embryonic fibroblasts (MEFs) and identified that most paternally imprinted genes were down-regulated and the maternally imprinted genes were up-regulated.
Project description:Haploid cells are amenable for genetic analysis because they contain only one set of chromosomes.Here,we report the derivation of haESCs from androgenetic blastocysts. These cells, which we designated AG-haESCs, express classical ESC markers, are pluripotent, and contribute to various tissues including the germline upon injection into diploid blastocysts. We used microarrays to compare the gene expression levels among androgenetic haploid embryonic stem cell lines(AG-haESC) E14 and male mouse embryonic fibroblasts (MEFs) and identified that most paternally imprinted genes were down-regulated and the maternally imprinted genes were up-regulated. To avoid the influence of diploidized cells on the expression profile, we collected samples from FACS of cells at G1/G0 stage by staining Hochest 33342. We used E14,which was a male embryonic stem cell lines, and MEFs isloated from male individuals as control. Gene expression profiles of all the cell lines were analysed on an Affymetrix GeneChip 430 2.0 array.
Project description:The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that usage of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.
Project description:The genomic DNA sample of AG-haESCs were compared to the C57BL/6J male mouse kidney by comparative genomic hybridization. The data confirmed that the haploid cells sustained genome integrity. The analysis was performed on a NimbleGen Mouse CGH 3x720K Whole-Genome Tiling Array to analyse the copy number variations in AG-haESCs, and the genomic DNA of C57BL/6J male mouse kidney was used as control, which had the same background with haploid ESCs.
Project description:Our lab first derived mouse androgenetic haploid embryonic stem cells (AG-haESCs) and demonstrated that AG-haESCs can be used as an “artificial spermatids” to generate gene-edited semi-cloned (SC) mice through intracytoplasmic injection (ICAHCI) into mature oocyte, even though the birth efficiency is very low. Further we proved that H19-DMR and IG-DMR were the main barrier to generate viable mice through androgenetic and parthenogenetic haESCs. More importantly, AG-haESCs mediated SC technology combined with CRISPR-Cas9 is a powerful tool to generate gene-modified mouse models and carry out genetic screening at organismal level. However, it is still not clear how the H19-DMR and IG-DMR coordinately regulate SC embryo development. Here, we found that the H19-DMR and IG-DMR regulate the development of SC embryos in spatio-temporal scales. Firstly, we found that the H19-DMR and IG-DMR are not indispensable for the development of preimplantation of SC embryos. Secondly, H19-DMR is essential for the development of SC embryos in mid-gestation and IG-DMR takes effect in late-gestation. Further, the maintenance of paternal H19-DMR methylation status and deletion of paternal H19 transcription unit play a key role in the structures and transport functions of SC embryo placenta. Importantly, AG-haESCs carrying triple deletions, including H19, H19-DMR and IG-DMR, can further improve the efficiency in generation of viable, normal-size, and fertile mice.
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.