Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.

Abstract

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (J3TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The cells produce both proinsulin I and II and efficiently process each into mature insulin, in a manner comparable to normal beta cells in isolated islets. Electron microscopy reveals typical beta-cell type secretory granules, in which insulin is stored. Insulin secretion is inducible up to 30-fold by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. (3TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype.Pancreatic beta cells synthesize and secrete insulin, a hormone involved in regulation of glucose homeostasis. In rodents there are two nonallelic insulin genes (I and II), which differ in the number of introns as well as in chromosomal location. Both genes are expressed in beta cells (1). An adult murine pancreas contains about 106 beta cells, clustered in the islets of Langerhans, which are dispersed throughout the exocrine tissue. As a consequence, molecular analyses of beta-cell function has in large part depended on in vitro cultures. Cells from isolated islets do not grow well in culture, although they maintain viability for a few weeks (2). In recent years, several lines of transformed beta cells have been generated (3-6). Two of these, RIN-m SF, derived from an x-ray-induced rat insulinoma, and HIT, from hamster islets transformed by simian virus 40, have been used extensively for characterization of insulin gene expression (4,5,7,8).However, it is unclear to what extent they represent normal beta cells, given that the levels of insulin secreted are considerably lower than those of beta cells in vivo.The ability to target expression of oncogenes to particular cells in transgenic mice, by using cell-specific regulatory elements, presents a method for immortalization of rare cell types. We have reported that transgenic mice harboring insulin-simian virus 40 tumor (T) antigen (RIP-Tag) hybrid genes heritably develop beta-cell tumors (9-11). Here we describe the characterization of several beta tumor cell (J3TC) lines obtained from transgenic mouse tumors and propagated in culture for over 60 passages. These cells provide a useful tool for studies of beta-cell regulation and gene expression. METHODSCell Cultures. Pancreatic insulinomas were excised from transgenic mice and disrupted in Dulbecco's modified Eagle's medium (DMEM). To minimize contamination by fibroblasts and other nontransformed cells, the tumors were not trypsinized. Rather, the tumor capsule was gently removed, and the tumor cells were mech...

Regulation of insulin preRNA splicing by glucose

“…A similar standard curve for mouse insulin II genomic DNA was used to convert the DNA data. This data can be used to estimate the number of insulin II RNA molecules per ␤TC3 cell at approximately 2 ϫ 10 4 , consistent with previous evidence that ␤TC3 cells maintain high insulin mRNA levels (21). The estimate of gene copy is 2 copies per cell, as expected for a diploid cell.…”

“…2B, the signals are compared from two mouse islet tumor cell lines: the insulin-producing ␤TC3 cells (21) and the glucagon-producing ␣TC1.6 cells (22). For the ␤TC3 cells, the light signal in the RNA assay increases linearly with the number of cells used, with the limit of detection at approximately one cell.…”

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

“…In these cases, insulinomas were produced from transgenic mice expressing an oncogene under the control of the insulin promoter allowing the expression of the transgene specifically in mature beta cells [24,51]. This approach was used to generate rodent beta cell lines that were functional in terms of insulin secretion upon glucose simulation.…”

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

“…The peculiar properties of CgB secretion could open a new line of functional research in beta cells, which remains to be developed. [21], RIN-5AH [22] and betaTC3 [23] cells were grown at 37°C, under a 5% CO 2 humidified atmosphere, in RPMI 1640 (INS-1E and betaTC3) or 11 mmol/l glucose-containing DMEM (RIN-5AH) media supplemented with 10% (vol./vol.) fetal clone serum III (Hyclone, Logan, UT, USA), 100 U/ml penicillin per streptomycin and 2 mmol/l ultra-glutamine (Cambrex, Verviers, Belgium).…”