Characterization of the Mouse Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein Gene Promoter by In Situ Footprinting

Abstract

Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (

Long-Range Enhancers Are Required to Maintain Expression of the Autoantigen Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein in Adult Mouse Islets In Vivo

“…Cell culture, transient transfection, and luciferase assays. Mouse pancreatic islet ␤-cell-derived ␤TC-3 cells were cultured and cotransfected with 0.5 g of an expression vector encoding Renilla luciferase (Promega) and 2 g of the indicated firefly luciferase plasmids using the lipofectamine reagent (Gibco BRL), as previously described (22). Following transfection, ␤TC-3 cells were harvested by trypsin digestion and then resuspended in passive lysis buffer (Promega).…”

“…Our work has focused on identifying the transcription factors that control IGRP gene expression with the goal of identifying novel, islet-enriched transcription factors important for pancreatic development and/or function (3,(21)(22)(23). We believe this approach is reasonable given that similar work (24 -26), focused on other islet-specific genes, has led to the identification of such proteins.…”

“…A key step in the identification of such factors is the definition of the minimal IGRP gene sequence necessary for mimicking the expression pattern of the endogenous IGRP gene. We have previously demonstrated that the Ϫ306 to ϩ3 region of the mouse IGRP promoter confers high-fusion gene expression in islet-derived cell lines (21)(22)(23)27) and is sufficient to initiate transgene expression in mouse islets, predominantly in ␤-cells, at the expected time in development, around embryonic day 14 (28). However, unlike the endogenous IGRP gene, expression of this transgene was markedly suppressed in adult mouse islets, suggesting that additional cis-acting elements in the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.…”

Insulin-mediated activation of activator protein-1 through the mitogen-activated protein kinase pathway stimulates collagenase-1 gene transcription in the MES 13 mesangial cell line

“…In one experiment (Fig. 8), attached MES 13 cells were co-transfected with a collagenase-CAT fusion gene construct (2 µg/ well) along with expression vectors encoding galactosidase (0·5 µg/well) and the insulin receptor (0·8 µg/well), using the lipofectamine reagent (Gibco-BRL, Invitrogen, Carlsbad, CA, USA) exactly as previously described (Bischof et al 2001). The lipofectamine:DNA ratio was 5:1.…”

“…Cells were grown in complete MsGM and re-plated the day before use into six-well culture plates. Attached cells were transiently co-transfected with collagenase-luciferase fusion gene constructs (14 µg/well) along with an expression vector encoding Renilla luciferase (0·7 µg/well) and either an expression vector encoding the insulin receptor or the empty pcDNA3 vector (2·8 µg/well), using the lipofectamine reagent (Gibco-BRL) exactly as previously described (Bischof et al 2001), except that the lipofectamine: DNA ratio was 0·7:1. After lipofectamine-mediated transfection, cells were then incubated for 18-24 h in basal MsGM prior to harvesting.…”