Novel insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase, and human insulin expression
| Autor: | Samuel A. Clark, P. Hansen, Sarah Ferber, Karl D. Normington, H. Constandy, Philippe A. Halban, Christian Quaade, Christopher B. Newgard |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Pancreatic Neoplasms/ genetics/pathology/secretion
Endocrinology Diabetes and Metabolism Cell Amylin RNA Messenger/analysis/genetics Polymerase Chain Reaction Glucokinase Insulin Secretion Insulinoma/ genetics/pathology/secretion Tumor Cells Cultured Insulin ddc:576.5 Transgenes Chromatography High Pressure Liquid Proinsulin Glucose Transporter Type 2 Plasmids/genetics Chemistry Transfection/genetics Glucokinase/ genetics Cell biology medicine.anatomical_structure Proinsulin/genetics/metabolism DNA Primers/chemistry Islets of Langerhans/metabolism Genetic Engineering Plasmids medicine.medical_specialty endocrine system Monosaccharide Transport Proteins Transgenes/genetics Transgene Transfection Islets of Langerhans Rats Nude Internal medicine medicine Internal Medicine Animals Humans RNA Messenger Insulinoma DNA Primers Monosaccharide Transport Proteins/ genetics medicine.disease Blotting Northern Rats Transplantation Pancreatic Neoplasms Endocrinology Gene Expression Regulation Cell culture Gene Expression Regulation/ genetics Insulin/analysis/ genetics/secretion Genetic Engineering/methods Protein Processing Post-Translational |
| Zdroj: | Diabetes, Vol. 46, No 6 (1997) pp. 958-967 Scopus-Elsevier |
| ISSN: | 0012-1797 |
| DOI: | 10.2337/diabetes.46.6.958 |
| Popis: | Cellular engineering studies in our group are directed at creating insulin-secreting cell lines that simulate the performance of the normal islet β-cell. The strategy described in this article involves the stepwise stable introduction of genes relevant to β-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term “iterative engineering.” RIN cells stably engineered to contain multiple copies of the human insulin gene exhibit a large increase in insulin content, such that they approach the content of human islets assayed in parallel. Analysis by high-performance liquid chromatography demonstrates that these engineered cell lines process human proinsulin to mature insulin with high efficiency. Cell lines that are further engineered to express the GLUT2 and glucokinase genes demonstrate stable expression of the three transgenes for the full lifetime of the lines produced to date (6 months to 1 year in continuous culture). Transplantation of the engineered cell lines into nude rats reveals that stably integrated genes are expressed at constant levels in the in vivo environment over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal β-cell, including rat insulin, amylin, sulfonyhirea receptor, and glucokinase, are stably expressed in the insulinoma lines during these in vivo studies. Endogenous GLUT2 expression, in contrast, is rapidly extinguished during in vivo passage. The loss of GLUT2 is overcome in engineered cell lines in which transporter expression is provided by a stably transfected trans gene. These results suggest that a potential advantage of the iterative engineering approach may be to preserve stability of function and phenotype, particularly in the in vivo setting. |
| Databáze: | OpenAIRE |
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