Suppressive Subtractive Hybridization (suppressive + subtractive_hybridization)

Distribution by Scientific Domains

Selected Abstracts

Expression of genes associated with allantois emergence in ovine and bovine conceptuses

A.M. Ledgard
Abstract In the development of ruminant embryos, the emergence and growth of the allantois is critical for the establishment of the chorioallantoic placenta. The allantoic membrane contributes to all the vasculature that perfuses the placental tissues and the fetal membranes. Using suppressive subtractive hybridization to compare mRNA from Day 13 ovine preimplantation conceptuses (prior to allantoic emergence) with Day 17 allantoic membrane, we identified nine genes whose expression was associated with the emergence of the allantoic sac. Collagen alpha 1 type XII, collagen alpha 2 type I, collagen alpha 2 type V, epsilon 4 beta-globin, osteonectin, and uroplakin were expressed at significantly greater levels in ovine Day 17 allantois compared to Day 13 conceptuses. These genes are associated with the extracellular matrix and most likely are involved in establishing and strengthening the structural integrity of the allantoic sac and in the development of the blood vessels. RalB expression increased with development although at significantly greater levels in the allantois only at Day 19. Hoxa-10 and RhoA showed no differential expression during this period. All these genes showed a similar temporal pattern of expression in bovine conceptuses at equivalent stages of development with significantly greater expression of all these genes, except for Hoxa-10, found in Day 24 allantois compared to Day 14 conceptuses. This suggests that the role they play in allantoic emergence, growth and function is conserved in both ruminant species and that their expression is regulated in a similar manner. The interactions and regulation of this process remains to be fully explained. Mol. Reprod. Dev. 1084,1093, 2006. 2006 Wiley-Liss, Inc. [source]

ORIGINAL ARTICLE: Calcineurin/NFAT Pathway: A Novel Regulator of Parturition

Chisa Tabata
Problem, The oxytocin (OT),oxytocin receptor (OTR) system plays an important role in mammalian parturition. However, we found OTR-deficient (OTRKO) mice are fertile and deliver at term without birth defects, thus alternative pathways inducing parturition can be hypothesized. Methods of study, We tested the gene expression profile of OTRKO mice using suppressive subtractive hybridization, and focused on the calcineurin/nuclear factor of activated T cells (NFAT) pathway. We examined the expression and localization of this pathway in mouse parturition. Results, Calcineurin and NFATc1 were detected in the decidua of pregnant uteri at term using immunohistochemistry (IHC). We identified higher activation levels of NFATc1 in wild type (WT) than in OTRKO mice and increased calcineurin A and NFATc1 mRNA levels during pregnancy. Moreover, injection of FK506, the inhibitor of this pathway, prolonged the delivery of the first pup. Conclusion, Our findings suggested that the calcineurin/NFAT pathway might play a substantial role in initiation of labor. [source]

An Immunomodulatory Role for Follistatin-Like 1 in Heart Allograft Transplantation

J. B. Le Luduec
Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8+ T cells and TGF, are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8+ T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFN,. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance. [source]

Dynamic changes in gene expression during vitellogenic stages of the white shrimp: Fenneropenaeus merguiensis de Man

Monwadee Wonglapsuwan
Abstract Ovarian maturation is a crucial step for shrimp brood stock. A suppressive subtractive hybridization was used to identify differentially expressed genes in the ovaries during vitellogenesis of Fenneropenaeus merguiensis. Three- to sevenfold up-regulated genes were selected. A blast search identified nine unique genes. The genes that may be involved in ovarian maturation, namely translationally controlled tumour protein (TCTP), heat shock protein 70 (HSP70), H-L(3)MBT-LIKE, shrimp ovarian peritrophin (SOP), vitellin (Vn), thrombospondin (TSP) and ribosomal protein L10a (RPL10a), were further studied. The transcripts of HSP70, TCTP, SOP and RPL10a in the ovary showed their highest expression in the early stage and declined in the later stages. In contrast, the transcripts of the H-L(3)MBT-LIKE, TSP and Vn genes increased from the early stage to be significantly up-regulated during the late stage. A comparison of gene expression among organs during the vitellogenesis showed that the transcripts of HSP70, SOP, H-L(3)MBT-LIKE and TSP were down-regulated in the brain, intestine, hepatopancreas and lymphoid (except for TSP) when compared with their expression in shrimp with non-developed ovaries. The mRNA of TCTP and RPL10a was significantly over-expressed in the lymphoid and heart, whereas TCTP transcripts were significantly down-regulated in the brain during the vitellogenesis. The molecular behaviour of the transcripts in this study may, in the future, lead to an ability to stimulate the ovarian development in shrimp. [source]

The putative-farnesoic acid O -methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre-imaginal life expression

Laura Vannini
Abstract Farnesoic acid O -methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative- FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real-Time PCR in medfly pre-imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre-imaginal putative- FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well-known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. 2010 Wiley Periodicals, Inc. [source]