NOVEL INSULINOMA CELL-LINES PRODUCED BY ITERATIVE ENGINEERING OF GLUT2, GLUCOKINASE, AND HUMAN INSULIN EXPRESSION

Cellular engineering studies in our group are directed at creating ins ulin-secreting cell lines that simulate the performance of the normal islet beta-cell. The strategy described in this article involves the s tepwise stable introduction of genes relevant to beta-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term ''i terative engineering.'' RIN cells stably engineered to contain multipl e 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 demons trates that these engineered cell lines process human proinsulin to ma ture insulin with high efficiency. Cell lines that are further enginee red to express the GLUT2 and glucokinase genes demonstrate stable expr ession of the three transgenes for the full lifetime of the lines prod uced to date (6 months to 1 year in continuous culture). Transplantati on of the engineered cell lines into nude rats reveals that stably int egrated genes are expressed at constant levels in the in vivo environm ent over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal beta-cells, including rat insuli n, amylin, sulfonylurea receptor, and glucokinase, are stably expresse d in the insulinoma lines during these in vivo studies. Endogenous GLU T2 expression, in contrast, is rapidly extinguished during in vivo pas sage. The loss of GLUT2 is overcome in engineered cell lines in which transporter expression is provided by a stably transfected transgene. These results suggest that a potential advantage of the iterative engi neering approach may be to preserve stability of function and phenotyp e, particularly in the in vivo setting.