The development of autologous somatic cells, engineered for the synthesis and release of human insulin under physiological stimuli, would certainly represent a major breakthrough in the therapy of insulin-dependent diabetes mellitus. We generated a retroviral vector containing the human proinsulin cDNA and the gene coding for the human nerve growth factor receptor for quantitative analysis of transduced cells. Primary rat hepatocytes were selected as target cells because of the constitutive expression of the pancreatic β-cell glucose transporter GLUT-2 and the glycolitic enzyme glucokinase. Appropriate conditions for culture and retroviral transduction are described. The highest transduction efficiency, evaluated as percentage of LNGFr expressing cells was obtained by repeated infection cycles (40±10%). Human proinsulin accumulated in the culture medium of transduced rat hepatocytes (mean±SD): 18.1±7.9 (range 8.7-36.4) ng/24h/10 6 cells. Primary rat hepatocytes can be efficiently transduced by a retroviral vector and the de novo synthesis of human proinsulin can be induced. Primary cultured hepatocytes represent an useful model to test retroviral constructs engineered for the glucose-inducible expression of insulin under the control of liver-specific promoters.
|Number of pages||4|
|Journal||Journal of Molecular Medicine|
|Publication status||Published - 1999|
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