Compensatory responses in mice carrying a null mutation for Ins1 or Ins2.

Intrauterine growth retardation and postnatal acute diabetes result from insulin deficiency in double homozygous null mutants for Ins1 and Ins2 (Duvillié B, et al., Proc. Natl. Acad. Sci. USA 94:5137-5140, 1997). The characterization of single homozygous null mutants for Ins1 or Ins2 is described here. Neither kind of mutant mice was diabetic. Immunocytochemical analysis of the islets showed normal distribution of the endocrine cells producing insulin, glucagon, somatostatin, or pancreatic polypeptide. Analysis of the expression of the functional insulin gene in Ins1-/- or Ins2-/- mice revealed a dramatic increase of Ins1 transcripts in Ins2-/- mutants. This compensatory response was quantitatively reflected by total pancreatic insulin content similar for both types of mutants and wild-type mice. Moreover, both mutants had normal plasma insulin levels and normal glucose tolerance tests. The determination of beta-cell mass by morphometry indicated beta-cell hyperplasia in the mutant mice. The beta-cell mass in Ins2-/- mice was increased almost threefold, which accounts for the increase of Ins1 transcripts in Ins2-/-mutants. This study thus contributes to evaluate the potential of increasing the beta-cell mass to compensate for low insulin production.

Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice.

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.

Human insulin gene in transgenic mouse lines.

Transgenic mouse lines carrying the human insulin gene flanked by 4 kilobases (kb) in 5' and 5.5 kb in 3' were obtained. The presence of the human C-peptide in serum and urine, and of specific transcripts of the human gene in pancreas RNA indicate that the human DNA fragment contains the sequences necessary for correct phenotypic expression of the gene.

B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice.

Transgenic mouse lines carrying the human insulin gene were previously shown to express it in pancreas but not in other tissues. The present study reports evidence that the expression of the transgene is restricted to a single category of cells. Immunofluorescence staining of frozen pancreas sections showed that the human C-peptide was present in pancreatic islets only, and more precisely in the B cells of the islets. Human insulin transcripts were initiated correctly in mouse pancreas at the same site as in human pancreas. Three different transgenic lines with different insertion sites and various copy numbers of the human insulin transgene had the same high levels of the transgene transcripts corresponding to a well-balanced contribution in insulin gene expression.

Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region.

A mini-human insulin gene and four derivatives mutated at several regions potentially involved in the regulation of gene expression were used to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied using 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 sections to examine whether expression of the transgene was still specifically expressed in beta-cells. Mutation of the cis-acting elements located 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 virus thymidine kinase gene (tk) promoter, failed to activate chloramphenicol acetyltransferase (CAT) gene expression in transfected insulinoma cells, while it was activated by the equivalent region of the rat insulin I gene. On the contrary, mutation of the DNA motifs located between -109 and -75 (GCI and CTI) or between -323 and -297 (CTIII) did not significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (-58/+l) by the tk promoter did not alter its level of expression in transgenic mice. In all instances, expression of the different transgenes remained localized in the islet beta-cells. Altogether, these results indicate that the GCII-CTII motif is an important regulatory element for efficient expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of other elements.

Genetic engineering in mice: impact on insulin signalling and action.

The expression of a number of genes encoding key players in insulin signalling and action, including insulin, insulin receptor (IR), downstream signalling molecules such as insulin receptor substrate-1 (IRS-1) and IRS-2, glucose transporters (GLUT4, GLUT2) and important metabolic enzymes such as glucokinase, has now been altered in transgenic or knockout mice. Such mice presented with phenotypes ranging from mild defects, revealing complementarity between key molecules or pathways, to severe diabetes with ketoacidosis and early postnatal death. Insulin action could also be improved by overproduction of proteins acting at regulatory steps. The development of diabetes by combining mutations, which alone do not lead to major metabolic alterations, validated the 'diabetogenes' concept of non-insulin-dependent diabetes mellitus. Genes encoding insulin-like growth factors (IGF-I and IGF-II) and their type I receptor (IGF-IR) have also been disrupted. It appears that although IR and IGF-IR are both capable of metabolic and mitogenic signalling, they are not fully redundant. However, IR could replace IGF-IR if efficiently activated by IGF-II. Studies with cell lines lacking IR or IGF-IR lend support to such conclusions. Concerning the issues of specificity and redundancy, studies with cell lines derived from IRS-1-deficient mice showed that IRS-1 and IRS-2 are also not completely interchangeable.

Pancreatic expression of human insulin gene in transgenic mice.

We have investigated the possibility of obtaining integration and expression of a native human gene in transgenic mice. An 11-kilobase (kb) human chromosomal DNA fragment including the insulin gene (1430 base pairs) was microinjected into fertilized mouse eggs. This fragment was present in the genomic DNA of several developing animals. One transgenic mouse and its progeny were analyzed for expression of the foreign gene. Synthesis and release of human insulin was revealed by detection of the human C-peptide in the plasma and urine. Human insulin mRNA was found in pancreas but not in other tissues. These findings indicate that the 11-kb human DNA fragment carries the sequences necessary for tissue-specific expression of the insulin gene and the human regulatory sequences react to homologous signals in the mouse.