Gene expression variation in horse placental and fetal tissue, and resting and stimulated horse lymphocytes [Agilent-018932]
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ABSTRACT: We undertook gene expression microarray experiments to identify genes that are differentially expressed in different placental and fetal tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, chorion, and fetal tissue. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a custom Agilent horse array designed in house that featured >14,000 probes on an 8x15k array format. Several genes were selected from the results for validation by quantitative real-time PCR. QPCR results matched the microarray results very closely. Four Day 33-35 chorionic girdle RNAs were compared to matching chorion RNAs, and fetal tissue from two of the conceptuses. Gene expression in resting lymphocytes was compared to gene expression in PWM treated lymphocytes.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in invasive (Chorionic Girdle) and non-invasive (Chorion) placental tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, and chorion. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format. Three day 33-35 chorionic girdle RNAs were compared to matching chorion RNAs. Gene expression in resting lymphocytes was compared to gene expression in PWM treated lymphocytes.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in invasive (Chorionic Girdle) and non-invasive (Chorion) placental tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, and chorion. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in different placental and fetal tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, chorion, and fetal tissue. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a custom Agilent horse array designed in house that featured >14,000 probes on an 8x15k array format. Several genes were selected from the results for validation by quantitative real-time PCR. QPCR results matched the microarray results very closely.
Project description:This SuperSeries is composed of the following subset Series: GSE35741: Gene expression variation in horse placental and fetal tissue, and resting and stimulated horse lymphocytes [Agilent-018932] GSE35742: Gene expression variation in horse placental tissue, and resting and stimulated horse lymphocytes [Agilent-021322] Refer to individual Series
Project description:In eutherian mammals, dosage compensation of X-linked genes is achieved by X chromosome inactivation. X inactivation is random in embryonic and adult tissues, but imprinted X inactivation (paternal X silencing) has been identified in the extraembryonic membranes of the mouse, rat, and cow. Few other species have been studied for this trait, and the data from studies of the human placenta have been discordant or inconclusive. Here, we quantify X inactivation using RNA sequencing of placental tissue from reciprocal hybrids of horse and donkey (mule and hinny). In placental tissue from the equid hybrids and the horse parent the allelic expression pattern was consistent with random X inactivation, and imprinted X inactivation can clearly be excluded. We characterized horse and donkey XIST gene, and demonstrated that XIST allelic expression in female hybrid placental and fetal tissues is negatively correlated with the other X-linked genes chromosome-wide, which is consistent with the XIST-mediated mechanism of X inactivation discovered previously in mice. As the most structurally and morphologically diverse organ in mammals, the placenta also appears to show diverse mechanisms for dosage compensation that may result in differences in conceptus development across species. Examine allelic expression from individual samples of invasive trophoblast tissue of the chorionic girdle from gestation day 33-34 conceptuses of 5 horses, 3 donkeys, 6 mules, and 1 hinny.
Project description:The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes of which 10 were paternally expressed. An additional 78 candidate novel imprinted genes identified by RNA-seq also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the novel genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase (HAT1), the first example of an imprinted gene involved in chromatin modification, and LY6G6C, the first imprinted gene to be identified in the major histocompatibility complex. The 78 novel candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting. Examine allelic expression from four individual samples of invasive trophoblast tissue of the chorionic girdle from gestation day 33 conceptuses of horse, donkey, mule and hinny.
Project description:The placenta is considered one of the candidate cell sources in cellular therapeutics because of a large number of cells and heterogenous cell population with myogenic potentials. We first analyzed myogenic potential of cells obtained from six parts of the placenta, i.e., umbilical cord, amniotic epithelium, amniotic mesoderm, chorionic plate, villous chorion (chorion frondosum), , and decidua basalis. Implantation of placenta-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of human dystrophin. Co-existence of human and murine nuclei in one myotube and presence of human dystrophin in murine myotube suggests that human dystrophin expression is due to cell fusion between host murine myocytes and implanted human cells. In vitro analysis revealed that cells derived from amniotic mesoderm, chorionic plate, ,and villous chorion efficiently transdifferentiate into myotubes. These cells fused to C2C12 murine myoblasts by in vitro co-culturing, and murine myoblasts start to express human dystrophin after fusion. These results demonstrate that placenta-derived cells, especially extraembryonic mesodermal cells, have a myogenic potential and regenerative capacity of skeletal muscle. Determination of cell specification with the gene chip analysis revealed that each placental cell has a distinct expression pattern. Experiment Overall Design: To isolate chorionic villi cells, we used the explant culture method, in which the cells were outgrown from pieces of chorionic villi attached to dishes. Chorionic villi cells were harvested with 0.25% trypsin and 1 mM EDTA, and overlaid onto the cultured fetal cardiomyocytes at 7 x 103/cm2. Every 2 days, the culture medium was replaced with fresh culture medium that was supplemented with 10% FBS and 1 ug/ml Amphotericin B (GIBCO). The morphology of the beating chorionic villi cells was evaluated under a fluorescent microscope.
Project description:We obtained placental issue between days 27 and 34 of pregnancy from matched mare and stallion pairs. We used whole transcriptome profiling in order to measure and compare gene expression in chorionic girdle trophoblast and adjacent regressing chorion at pregnancy day 27 (initiation of proliferation and prior to differentiation), day 30 (initiation of differentiation), day 31 (consolidation of differentiation and movement of cells) and day 34 (when the majority of the trophoblast cells have terminally differentiated into binucleate eCG-secreting trophoblast and have started to obtain invasive qualities and immunomodulatory capacities). Differentially expressed genes were then identified to determine functions and signalling pathways whose activity was modulated over this critical period of trophoblast development. A selection of genes and pathways were subsequently validated.