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Bovine leukosis --- Virology --- gene expression
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Bovine leukosis --- viruses. --- viruses --- genetic code --- cloning --- genetic engineering --- computer applications --- Logiciel blast
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Exostosin is a family of type II transmembrane glycosyltransferase enzymes, containing five members: EXT1, EXT2, EXTL1, EXTL2, EXTL3. These proteins are linked with heparan sulfate chains polymerization on heparan sulfate proteoglycans, but recent advances demonstrated that some of these members exhibit novel functions. Rab GTPases family is composed of at least 60 members of membrane-bound proteins that regulate many steps of membrane trafficking. Recent studies showed that when EXT1 is depleted and Rab10 overexpressed in human cells, NOTCH1 expression was enhanced. EXT1 silencing also induces major morphological changes in the cell, particularly in the endoplasmic reticulum and the Golgi apparatus. The aim of this work is to investigate members of EXT and Rab GTPases families to determine the specificity of the results obtained for EXT1 and Rab10 using NOTCH1 as a reporter. Rab GTPases were cloned in an expression vector and transfected in human cells to observe their potential effect on NOTCH1 expression. Cell lines silenced for each EXT member were created and their characteristics assessed. EXT1 and EXT2 protein production in Yarrowia lipolytica was also considered to perform in vitro analyzes using purified proteins. As a result, a functional library of Rab GTPases in expression vector was created, verified, and is ready to be used for transfection experiments. Stable knockdown cell lines were successfully created for EXT genes, a reduction of NOTCH1 expression was observed when EXT2 or EXTL3 were silenced, probably due to modification of the trafficking mechanism and/or genetic co-regulation. Protein production did not show significant results. These results suggest a new model for increased NOTCH1 trafficking: overexpression of EXT2 and Rab10 with simultaneous silencing of EXT1.
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viruses. --- lymphocytes --- transport. --- Glucose --- proteins --- Immunogenetics
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