433 NEW TRANSGENIC PIG DIABETES MODEL EXPRESSING THE MUTANT Insulinc93S FOR THE STUDY OF THE PANCREATIC BETA CELL DYSFUNCTION

We previously established a mutant mouse line showing diabetes which was caused by a point mutation in the Insulin 2 (Ins2) gene. The point mutation leads to the amino acid exchange C95S and the loss of the A6-A11 intrachain disulfide bond of insulin. Male heterozygous Ins2C95S mutant mice develop progressive diabetes mellitus with strong reduction of the total pancreatic islet volume and the total beta cell volume together with severe alterations of the beta cell structure. As pigs share many anatomical and physiological characteristics with humans, we aimed to establish a transgenic pig model expressing the mutant insulin by additive gene transfer for the subsequent study of beta cell dysfunction in diabetes mellitus. The transgene consisted of German Landrace insulin sequences including 1.3 kb of the insulin promoter and 1 kb insulin gene sequences with the 3 exons and the T to A (C93S) point mutation in exon 3 analogous to the mutant mouse insulin. A floxed neomycin resistance gene at the 3′ end of the transgene was used as a selection marker. After successful transfection of male fetal fibroblasts of both the German Landrace and the Schwaebisch- Haellisch pig breeds, pooled transgenic fibroblasts were used for somatic cell nuclear transfer (SCNT). Five hundred three reconstructed pig embryos were generated and endoscopically transferred to 5 synchronized recipients. One pregnancy with German Landrace genetic background and one pregnancy with Schwaebisch-Haellisch genetic background of the cloned embryos developed to term, which gave rise to 3 living offspring from each of the 2 pregnancies; 5 of the 6 piglets were transgenic. Southern blot analysis showed different transgene signal patterns in all animals examined. Transgenic pigs of both litters revealed unaltered fasting blood glucose levels up to an age of 8 months. However, disturbed intravenous glucose tolerance and reduced insulin secretion were detected in 1 transgenic pig of the first litter at 8 months of age. The area under the glucose curve of this transgenic pig was 75% larger (22 136 v. 12617) and the area under the insulin curve 53% smaller (1250 v. 2670) compared with the control. Ongoing analyses comprise glucose tolerance tests in the second litter as well as pathohistologic analysis of the pancreata of both litters. Cells from suitable transgenic founders will be used for recloning to establish a new transgenic pig model expressing the mutant InsulinC93S for an in-depth study of pancreatic beta cell dysfunction in diabetes mellitus. Supported by the Deutsche Forschungsgemeinschaft (GRK1029).