HUMAN INSULIN GENE-EXPRESSION IN TRANSGENIC MICE - MUTATIONAL ANALYSIS OF THE REGULATORY REGION

A mini-human insulin gene and four derivatives mutated at several regi ons potentially involved in the regulation of gene expression were use d to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied us ing three approaches: (1) Northern blot analysis using total RNA from pancreas and other organs, (2) radioimmunoassay to detect the human C- peptide in urine samples, and (3) immunocytochemistry of pancreas sect ions to examine whether expression of the transgene was still specific ally expressed in beta-cells. Mutation of the cis-acting elements loca ted between -238 and -206 (GCII and CTII motifs) resulted in a strong decrease of gene expression in the pancreas of transgenic mice, but it did not lead to complete extinction of the transgene expression. This region alone (-255/-202), when linked to the minimal Herpes simplex v irus thymidine kinase gene (tk) promoter, failed to activate chloramph enicol acetyltransferase (CAT) gene expression in transfected insulino ma cells, while it was activated by the equivalent region of the rat i nsulin I gene. On the contrary, mutation of the DNA motifs located bet ween -109 and -75 (GCI and CTI) or between -323 and 297 (CTIII) did no t significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (-58/+1) by th e tk promoter did not alter its level of expression in transgenic mice . In all instances, expression of the different transgenes remained lo calized in the islet beta-cells. Altogether, these results indicate th at the GCII-CTII motif is an important regulatory element for efficien t expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of othe r elements.