2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

CELL AND DEVELOPMENTAL BIOLOGYIdentification of Integrins Expressed on Apical Membranes of Endometrial and Trophectoderm Cells ofPigs during Early PregnancyWendy Browning*, Dana Massuto, Laurie JaegerDepartment of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences,Texas A&M UniversityMany embryonic losses occur in domestic pigs and other mammals during the time of implantation and placentation;a better understanding of the mechanisms underlying these processes is needed to prevent these losses. The objective of thisstudy is to identify integrin proteins found on the luminal surface of the porcine uterus and conceptus trophectoderm duringearly pregnancy. Integrins are candidates for binding to extracellular matrix proteins such as the beta transforming growthfactor’s (TGF-beta) latency associated peptide (LAP). Interactions between integrins and LAP have been implicated inadhesion and signaling between the endometrium and conceptus trophectoderm during early pregnancy.Immunoprecipitation (IP) is being used to pull down the integrins from cell-surface labeled (biotinylated) culturedtrophectoderm cells (Tr2) as well as from similarly labeled endometrium. Detection of the proteins with Avidin-HorseradishPeroxidase (AHRP) is used to identify the integrins as surface proteins. Definitive identification will be based on relativemolecular mass and western blotting using rabbit and mouse antibodies that recognize specific integrins. Identification ofthese integrins will provide the basis for in depth study of their interactions with LAP at the conceptus-maternal interface inpigs and other mammals, including humans.Evaluation of DNMT-1 knockdown in bovine fetal fibroblasts using RNA interference (RNAi)Catherine Clinton*, Todd Stroud, Charles Long and Mark WesthusinDepartment of Veterinary Physiology and Pharmacology, College of Veterinary Medicine Texas A&MUniversity, College Station, TexasEfforts to derive cloned animals by somatic cell nuclear transfer result in a high rate of embryonic and fetalmortality. One theory for these problems is that during the epigenetic reprogramming phase of preimplantation development,the controlled expression of DNA Methyltransferase-1 (DNMT-1) is lost. Proper expression of DNMT-1 is essential forestablishment of the epigenetic code and conserving imprinted gene expression during the pre-implantation development.By using RNA-interference (RNAi) to knock down DNMT-1, we will attempt to artificially control DNMT-1 expression inbovine fetal fibroblasts (BFF) prior to their utilization for somatic cell nuclear transfer. We hypothesize this will result in anincrease in the percentage of cloned embryos that develop normally. To test this hypothesis, BFFs were transfected with ashort hairpin RNA (shRNA) designed to silence the expression of DNMT-1, using a lentiviral vector with green fluorescentprotein (GFP) as a reporter gene. In order to evaluate and quantify DNMT-1 expression and function, normal and transgenicBFF cells will be labeled for DNMT-1 and 5-methyl cytosine (5MeC) using immunocytochemistry (ICC). It is predicted thatthat transgenic BFF cells will have a decreased amount of DNMT-1 protein and reduced 5MeC (indicative of decrease inchromatin methylation) in comparison to normal BFF cells. Quantitative analysis of DNMT-1 and 5MeC will be conductedusing NIS Elements Software. The use of transient RNAi to knockdown DNMT-1 expression in cells prior to cloning maybe a useful tool that can be applied to correct aberrant reprogramming that occurs in preimplantation cloned embryos.3-O-Sulfotransferase (3OST) Role in Zebrafish EmbryogenesisKim Dean, Ken Krammer PhD, Josh DeweeuwNational Institute of Health - National Heart, Lung and Blood InstituteProteoglycans are ubiquitous macromolecules found throughout the body and participate in cellular functions suchas cell-cell communication and signal transduction. These molecules are composed of a single core protein and carbohydratechains known as glycosaminoglycans. Heparan sulfate(HS) is a glycosaminoglycan component of proteoglycans widelyknown for its ability to bind antithrombin, together inactivating thrombin and thereby blocking an essential step in the bloodcoagulation cascade. HS is also pivotal for cell-cell communication during embryogenesis of Brachydanio rerio,commonly known as zebrafish. In the zebrafish development model, sulfate group additions can be found on various carbonsof the alternating glucuronic acid (GlcA) and N-acetylglucosamine (GluNAc) disaccharide units of HS. Studies have shownthat adding a sulfate group to the 3 carbon of GluNAc by the enzyme 3-O-Sulfotransferase (3OST) represents less than half apercent of the modifications made to the GAG chain, yet many isoforms or genes regulate this modification. In contrast,sulfate groups added to the 2 carbon of the GlcA occurs in over 50% of all modifications and yet is regulated by a singleisoform of 2OST. 3OST?s seemingly paradoxical quantity of genes, despite low occurrences of sulfate groups at the 3carbonof GluNAc, perhaps reflects an important role in development. One of the goals of this project is to observe the events thatlead to the common phenotype seen in zebrafish embryos with reduced 3OST5, development ceasing prior to gastrulation.Recording the events that lead to this phenotype will help elucidate the cause of the defect by studying the movement and143