Expression data from rat androgenetic embryonic stem cells
ABSTRACT: 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: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:We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse?rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse?rat ES chimera system. The mouse?rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.
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:Induced pluripotent stem (IPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. The majority of prior established rat IPS cells were generated from somatic cells by retroviral and lentiviral transduction with expression of Oct4, Sox2, Klf4 and c-Myc and using chemical inhibitors of key differentiation pathways. A major difficulty in the application of this technology is the efficient delivery of reprogramming factors and the long-term maintenance of properties of stem cells. Here, we employed the PiggyBac (PB) transposon carrying four 2A peptide-linked reprogramming factors for generating rat IPS cells. These stable rat IPS cells are similar to embryonic stem (ES) cells in morphology, proliferation, teratoma formation, expression characteristic pluripotency markers, developmental potential, and germline transmission. Transcriptional profiling of the IPS cells revealed both pathways in common with ES cells from rat and unique signaling pathway to our cells, including Wnt, TGF and Notch. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying germline pluripotency and pave the way for exploration of cell-based therapies using the rat. To compare the gene expression profiling between rat IPS cells and ES cells to show if the rat IPS cells had been reprogrammed into pluripotent status like rat ES cells at the gene expression level.
Project description:In most embryos, the mid-blastula transition is a complex process featuring maternal RNA degradation, cell cycle pause, zygotic transcriptional activation and morphological changes. The nucleocytoplasmic (N/C) ratio has been proposed to control the multiple events at MBT. To understand the global transcriptional response to the changes of the N/C ratio, we profiled wild type and haploid embryos using cDNA microarrays at three developmental stages. Experiment Overall Design: For the diploid transcriptional profile, we prepared cDNA from hand selected wild-type embryos at cycle 13 interphase (15min after the nuclear division of cycle 12) and at early and late cycle 14 interphase (15min and 40 min after the nuclear division of cycle 13), respectively. For the haploid, cDNA was prepared from cycle 14 embryos (15min after the nuclear division of cycle 13) and early and late cycle 15 embryos (15min and 40 minutes after the nuclear division of cycle 14). The collection was designed in this way so that each cell cycle stage of the diploid has a counterpart stage with matched N/C ratio in the haploid (e.g. cycle 13 in the diploid has the same N/C ratio as cycle 14 in the haploid). In all experiments, the cell cycle progression was monitored by the Histone-GFP pattern. Triplicates of about 50 embryos each with right age were collected for each time point.
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:gene expression differences were analysed between haploid and diploid ES cells biological triplicates of haploid and diploid ES cell lines were analysed on an Affymetrix GeneChip 430 array
Project description:This SuperSeries is composed of the following subset Series: GSE30744: Expression analysis of haploid and diploid ES cells in 2i medium GSE30749: CGH analysis of haploid ES cells Refer to individual Series
Project description:Haploid mammalian embryonic stem cells (ESCs) hold great promise for functional genetic studies and assisted reproduction. Recently, rodent androgenetic haploid ESCs (AG-haESCs) were generated from androgenetic blastocysts and functioned like sperm to produce viable offspring via the intracytoplasmic AG-haESCs injection into oocytes. However, the efficiency of this reproduction was very low. Most pups were growth-retarded and died shortly after birth, which is not practical for producing knockout animals. Further investigation suggested a possible link between the low birthrate and aberrant expression of imprinted genes. Here, we report the high-frequency generation of healthy, fertile mice from H19-Igf2 imprinting-locus modified AG-haESCs, which maintained normal paternal imprinting and pluripotency. Moreover, it is feasible to perform further genetic manipulations in these AG-haESCs. Our study provides a reliable and efficient tool to rapidly produce gene-modified mouse models and will benefit reproductive medicine in the future.
Project description:The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models. Application of this technology to engineer genes in rats has not previously been possible because of the absence of germline-competent ES cells in this species. We have recently established authentic rat ES cells. Here we report the generation of gene knockout rats using the ES-cell-based gene targeting technology. We designed a targeting vector to disrupt the tumour suppressor gene p53 (also known as Tp53) in rat ES cells by means of homologous recombination. p53 gene-targeted rat ES cells can be routinely generated. Furthermore, the p53 gene-targeted mutation in the rat ES-cell genome can transmit through the germ line via ES-cell rat chimaeras to create p53 gene knockout rats. The rat is the most widely used animal model in biological research. The establishment of gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provide a powerful new platform for the study of human disease.