Gene expression data from ahES cells, ES cells, MEF cells and round sperm
ABSTRACT: Haploid stem cells offer an easy-to-manipulate genetic system and therefore have great values for studies of recessive phenotypes. Here, we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into 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 germline of chimeras 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. We used microarrays to compare the global programme of gene expression among ahES cells, normal diploid ES cells, MEF cells and round sperm cells and found that gene expression pattern of ahES cells was highly similar with ES cells but was distinct from MEF cells and round sperms. Androgenetic haploid ES cells were FACS sorted to harvest the G0/G1 phase haploid cells. Total RNA were extracted from three ahES cell lines (AH129-5, AH129-N1, AH129-NC1, all 129Sv genetic background), two ES cell lines ( CS1-1, R1, 129Sv background), MEF cells and round sperm and hybridized with Affymetrix GeneChip 430 2.0 array. Data were collected and analyzed to compare their gene expression pattern.
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:Generation of haploid gametes in vitro can potentially address gamete failure-based infertility.This study reports complete in vitro meiosis from murine ESC-derived PGCLCs resulting in the formation of male spermatid-like cells (SLCs) capable of producing viable fertile offspring via intracytoplasmic sperm injection (ICSI).Our findings provide the basis for generation of haploid spermatids in vitro in human, the generation of transgenic animals, and the use of this system to investigate mechanisms of meiosis. We used microarrays to compare gene expression profiles of in vivo and in vitro derived PGC cells and round spermatids. We collected E12.5 male fatal PGCs, PGCLC in vitro, round spermatids and spermatids like cells produced in vitro, each sample has 3 replications.
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 rat androgenetic embryonic stem cells (RahES cells) have only 21 chromosomes. However, they express pluripotency markers, differentiate into three germ layer cells as well as contribute to the germline as the normal diploid rat ES cells. Moreover, the RahES cells can produce fertile rats after intracytoplasmic injection into oocytes, thus are capable to transmit genetic modifications to offspring. We used microarrays to detail the global gene expression profile of RahES cells and identified distinct classes of up-regulated and down-regulated genes compared with the expression of normal diploid rat ES cells. Two different RahES cell lines, one diploid rat ES cell line and round sperm cells were selected for RNA extraction and hybridization on Affymetrix Chip.
Project description:Uniparental reproduction is widespread among lower vertebrates, but not in mammals. Researchers have produced bimaternal mice with a deletion of the H19 imprinted region. However, the mechanism of a single deletion in an immature oocyte sufficient to cross uniparental barriers is unknown. We found bimaternal-derived mice to be defective in multiple aspects. More importantly, bipaternal reproduction has not been achieved in mammals. Thus, the barrier of uniparental reproduction in mammals has not been elucidated. We identified a DNA hypomethylation status in haploid embryonic stem cells similar to that in primordial germ cells, with which bimaternal and bipaternal mice can be obtained by injection of haploid ES cells with genetic modifications. The phenotypic analyses of derived mice support the genetic conflict theory of genomic imprinting. Taken together, our results highlight the factors necessary for crossing uniparental reproduction barriers in mammals. Overall design: Reduced representation bisulfite sequencing (RRBS) for a global DNA methylation analysis of wild type (WT) or the differentially methylated region knocking out (DMR-KO) haploid ESCs androgenetic haploid ESCs (ahESC), parthenogenetic haploid ESCs (phESCs), diploid ESCs, embryos and brain tissue. Compared to wild type (WT) embryo, which was produced by normal zygote deriving from normal sperm and oocyte, the bimaternal (BM) embryo was produced through injecting a phESC into an MII oocyte and the bipaternal (BP) embryo was produced through injecting injecting an ahESC and a sperm into an enucleated oocyte.
Project description:The genomic DNA sample of monkey PG-haESCs were compared to the female adipose cells by comparative genomic hybridization. The data confirmed that these haploid cells sustained genome integrity. The analysis was performed on a Agilent aCGH G3 Rhesus Macaque 4x180K array to analyse the copy number variations in monkey PG-haESCs, and the genomic DNA of femal monkey adipose was used as control, which had the same background with haploid ESCs.
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 monkey parthenogenic blastocysts. These cells, which we designated PG-haESCs (parthenogenic haploid embryonic stem cells), express classical ESC markers, are pluripotent, and can differentiate to different cell lines from all three embryonic germ layers in vivo and in vitro. We used microarrays to compare the gene expression levels among PG-haESC, ICSI-derived ESCs and female monkey somatic fibroblasts. We used ICSI-derived ESCs and somatic fibroblasts isloated from female individuals as control. Gene expression profiles of all the cell lines were analysed on an Affymetrix Rhesus Macaque array.
Project description:To further understand the mechanism of reprogramming, the mouse embryonic fibroblast cells were infected with Oct4, Klf4, c-Myc and Sox2 with special sequence. 1) Oct4 and Klf4 containing retrovirus was delivered on day 0. 2) Oct4, Klf4 and c-Myc were delivered on Day 1.5. 3) c-Myc and Sox-2 were deivered on Day 3. 4) Sox2 were delivered on Day 4.5. 5) Vitamin C containging medium were used from Day 6. The gene expression of Day 0 (MEF), Day 1.5, Day 3, Day 4.5 and Day 6 were analyzed with micro array to indentify the possible underlying mechanism. Especially the factors related to MET/EMT, cell cycle and epigenetic were focused. MEF cells were subjected for reprogramming with special protoocl: 1) Oct4 and Klf4 containing retrovirus were delivered on day 0. 2) Oct4, Klf4 and c-Myc were delivered on Day 1.5. 3) c-Myc and Sox-2 were deivered on Day 3. 4) Sox2 were delivered on Day 4.5. 5) Vitamin C containging medium were used from Day 6. RNA on Day 0 (MEF), Day 1.5, Day 3, Day 4.5 and Day 6 were collected for analysis.