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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). [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF), the most common inherited disease in the Caucasian population, is caused by mutations of the CFTR gene, coding for an anion channel responsible for chloride movement, determining transepithelial salt transport, fluid flow and ion concentrations. Cystic fibrosis is determined by chronic lung disease, marked by airway surface dehydration, reduced mucus clearance and increased mucus obstruction, impaired bacterial killing and inflammation and continuous destruction of the lung tissue. The lack of suitable CF mouse models has so far impeded the understanding of disease mechanisms and the development of novel therapies. Recently, a pig model reflecting the main hallmarks of the human disease in the respiratory, gastrointestinal and genital tracts has been reported (Rogers et al. 2008 Science 321, 1837-1841). In the present study, we generated a new CF pig model by disrupting the CFTR gene using an approach that differs in 3 major aspects from the previously published model: (1) gene targeting was achieved using a modified bacterial artificial chromosome (BAC) vector; (2) transcription of the CFTR gene was completely prevented; and (3) homozygous CFTR mutant pigs were derived by nuclear transfer from cells in which both alleles were inactivated by sequential BAC targeting. We characterised 2 cloned litters with a total of 13 piglets (including 4 stillborns). All piglets showed a characteristic CF multi-organ disease with severe meconium ileus, microcolon, microgallbladder, hypoplasia of the exocrine pancreas, congenital aplasia of the vas deferens and malformations of the trachea. In contrast to the recently published CF pig, where the majority of the intestinal obstructions occurred proximal to the ileocecal junction, we found obstructions exclusively distal to this site. We thus present a novel pig model for CF that reflects the main hallmarks of the human disease. It confirms the suitability of the CFTR mutant pig for cystic fibrosis research and increases the availability of CF models for molecular and translational CF research. The help by Prof. K. Heinritzi and his team from the Pig Clinic, LMU Munich, is gratefully acknowledged. This study was supported by Mukoviszidose e.V. [ABSTRACT FROM AUTHOR]

Generation of transgenic pigs by somatic cell nuclear transfer (SCNT) has provided a new avenue for medical research. However, low efficiency of SCNT greatly hinders its practical applications. In this study we evaluated the efficiency of producing cloned transgenic pigs as well as the side effects of cloning observed in several piglets. As nuclear donors, we used 31 different cell cultures of porcine fetal fibroblasts, kidney cells, or skin fibroblasts derived from commercial breeds. Donor cells were modified by additive gene transfer or knockout with 21 different gene constructs or genes of interest. For additive gene transfer, a mixed cell population without previous expression analysis was used. For re-cloning, kidney or skin fibroblasts from cloned transgenic pigs were used. SCNT was performed using in vitromatured oocytes as previously reported (Kurome et al.2006 Transgenic Res. 15, 229–240). Reconstructed oocytes were transferred laparoscopically to oestrus synchronized recipient gilts. After transfer of 9317 reconstructed oocytes into 112 recipients, 58% of recipients became pregnant and 42% of them gave birth to offspring. Birth was induced by injection of a PGF2α-analogon. Out of 159 cloned piglets, 65 (40%) were clinically healthy and developed further normally, whereas 35 (22%) cloned piglets were stillborn and 46 (30%) died soon after birth. The major reason of early neonatal death was severe underweight (<700g), which has been found in 26 cases of several cloned litters. In rare cases, we also observed malformations like oversized tongue, cleft palate, or atresia ani. Thirteen piglets were lost because of other circumstances (killed by mother or infectious disease). The in vivodevelopmental competence of donor cells was analysed regarding the effects of genomic modification and re-cloning. Interestingly, we did not find significant differences in the pregnancy and delivery rates when using transfected and non-transfected cells. While the litter size from transfected cells was slightly smaller compared with non-transfected cells, the ratio of piglets per litter which survived the critical neonatal age was similar, namely 1.8 from transfected and 2.0 from non-transfected cells. Compared to the data from cloning, re-cloning resulted in lower pregnancy and delivery rates. Furthermore, efficiency with respect to the litter size or the number of clinically healthy piglets was significantly lower, as we obtained only 6 piglets from 4 pregnancies and 5 of them died at the neonatal age. In conclusion, cloned transgenic pigs were produced with several types of donor cells and genetic modifications. Nearly half of them showed abnormalities, which were not associated with the type of donor cells or the transgene used. Cloning efficiency was not affected by the procedure of genomic modification but was negatively influenced by re-cloning probably because of epigenetic alterations in the genome of donor cells. Supported by the DFG (FOR535, FOR793), by the Bayerische Forschungsstiftung, and by Mukoviszidose e.V. [ABSTRACT FROM AUTHOR]

The development of a1,3-galactosyltransferase deficient pigs along with overexpression of complement regulatory proteins was an important step to overcome hyperacute rejection of pig organs transplanted into primates. However, the lack of galactose-α1,3-galactose (αGal) epitopes does not protect against subsequent rejection mechanisms, such as acute vascular rejection, which plays a crucial role to achieve prolonged graft survival in pig-to-human xenotransplantation. Acute vascular rejection involves incompatibilities between human blood and porcine endothelial cells, leading to coagulation disorders. Indeed, thrombotic microangiopathy is one of the key characteristics of rejected pig-to-primate grafts from aGal-deficient donor animals. One of the strategies to overcome this coagulation problem by genetic modification of donor pigs is expression of human thrombomodulin (TM) on the porcine endothelium. Although porcine TM is able to bind circulating human thrombin, the resulting heterodimer is unable to cleave human protein C, which promotes the coagulation cascade. We addressed this problem by the development of a vector placing the single-exon open reading frame of human TM under the control of a 6-kb promoter fragment obtained from the porcine TM gene. In addition, we replaced the 3′-untranslated region (UTR) by a polyadenylation box from the bovine growth hormone gene to avoid interferon-y-dependent destabilization of thrombomodulin transcripts. This vector was linked to a floxed neomycin cassette and transfected into porcine fetal fibroblasts. Positive cell clones were selected with G418, mixed, and then used for nuclear transfer. Donor fibroblasts were electrofused with enucleated oocytes resulting in 358 reconstructed embryos (91.1% fusion rate). One or two days later, 294 embryos were transferred to 3 synchronized gilts. In total, 8 cloned transgenic piglets with 1 or 2 independent integration sites of the transgene were born. Heart, kidney, lung, and liver of 5 founder animals were analyzed by immunohistochemistry They were found to express thrombomodulin strongly and strictly limited to the vascular endothelium of at least heart and kidney. The staining intensity was estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Cells from founder pigs expressing TM strongly have been used for recloning. The effect of human TM to normalize blood coagulation will be tested in experiments involving ex vivoperfusion of transgenic hearts and kidneys with human blood. Supported by the Deutsche Forschungsgemeinschaft (FOR 535). [ABSTRACT FROM AUTHOR]

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted in response to nutrients and enhance glucose-induced insulin secretion. The insulinotropic action of GIP is impaired in type 2 diabetes (T2D) whereas that of GLP-1 is preserved. To evaluate the role of an impaired GIP function in the pathogenesis of T2D in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in the pancreatic islets. GIPRdn transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the pancreas were performed in 3 different age groups to investigate the effects of an impaired insulinotropic action of GIP on glucose metabolism and pancreas morphology. The insulinotropic action of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in 11-week-old GIPRdn transgenic pigs compared with control pigs. Eleven-week-old GIPRdn transgenic pigs (n= 5) exhibited significantly reduced oral glucose tolerance (P< 0.05) with a delay in insulin secretion compared with controls (n= 5). The area under the insulin curve (AUC) during the first 45 min following glucose load was 31% smaller (P< 0.05) in transgenic pigs compared with controls. The total insulin secretion capacity was not different between the 2 groups indicating that GIPRdn expression initially only interferes with the incretin effect. This was supported by the fact that intravenous glucose tolerance and insulin secretion in transgenic pigs were not different from controls. Five-month-old GIPRdn transgenic pigs revealed markedly reduced insulin secretion in response to oral glucose challenge (P< 0.01), resulting in significantly elevated glucose levels (P< 0.05). Also, intravenous glucose tolerance and insulin secretion were diminished in 11-month-old transgenic pigs. To determine the reason for the alterations in glucose metabolism, quantitative-stereological analyses of the pancreas were performed. In 11-week-old pigs, transgenic and control groups showed similar β-cell mass (n= 5 in each group). However, pancreatic β-cell mass was reduced by almost 40% (P< 0.05) in 5-month-old pigs and by 60% (P< 0.01) in adult (1 to 1.4 years) GIPRdn transgenic pigs compared with controls. To investigate the reason for the progressive reduction of pancreatic β-cell mass in GIPRdn transgenic pigs, β-cell proliferation rate was determined performing a double-immunohistochemistry for insulin and the proliferation marker Ki67. Eleven-week-old GIPRdn transgenic pigs showed significantly fewer Ki67-positive cell nuclei compared with controls (P< 0.05). In conclusion, GIPRdn transgenic pigs exhibit a comparable situation to T2D, such as impaired insulinotropic action of GIP, disturbed oral and intravenous glucose tolerance, and progressive reduction of β-cell mass. These alterations are at least partly attributable to diminished proliferation of β-cells. Grant support: Deutsche Forschungsgemeinschaft (GRK 1029), Bayerische Forschungsstiftung (492/02). [ABSTRACT FROM AUTHOR]

Among the candidate organs or tissues for pig-to-primate xenotransplantation, pancreatic islets are probably closest to clinical application. Rejection of islet xenografts occurs mainly by cellular mechanisms; that is, T cells. A candidate molecule to protect porcine islets against the attack by human T cells is CTLA-4Ig, which represents the T-cell-inactivating extracellular domain of the human CTLA-4 protein linked to a region of the human immunoglobulin (Ig). This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T-cell proliferation and T-cell-dependent antibody production. The survival of human, rabbit, and porcine islets after transplantation into streptozotocin-treated diabetic mice was found to be prolonged after treatment with CTLA-4Ig. In order to facilitate local protection of pig-to-primate islet xenografts, we generated transgenic pigs expressing LEA29Y, a modification of the original CTLA-4Ig with higher potency, specifically in the pancreatic islets. In LEA29Y, 2 amino acids in the binding region of CTLA-4 are altered. The LEA29Y coding sequence was placed under the control of the 1.3-kb core promoter from the porcine insulin gene (INS), and the polyadenylation signal from the bovine growth hormone gene (GH) was added. The construct was linked with a floxed neomycin resistance cassette and transfected into porcine fetal fibroblasts. The cells were selected and stable clones were pooled and used as donors for nuclear transfer. After electrofusion and activation, embryos were transferred to 2 synchronized gilts; 8 piglets survived to term with 7 of them carrying the transgene. Southern blot analysis suggested that the founder animals contain 1 or 2 independent integration sites. Four founders were autopsied at the age of 3 months to evaluate expression of LEA29Y in the pancreatic islets by immunohistochemistry. The ratio of immunohistochemically stained islet cell profiles to all islet cell profiles in the islet profiles visible in the sections was estimated. The staining intensity was also estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Although 2 founders exhibited single LEA29Y-positive islet cells in some pancreatic islet profiles, the other 2 founders showed a high percentage of strongly positive cells in all islet profiles examined, suggesting beta-cell specific expression. Fibroblasts from the latter 2 founders are currently being used for recloning to generate multiple pigs with constitutive expression of LEA29Y in the pancreatic islets. The protective effect of this strategy will be tested by transplanting LEA29Y-expressing porcine islets in diabetic mouse models with a humanized immune system and in diabetic nonhuman primate models. Supported by the Deutsche Forschungsgemeinschaft (FOR 535). [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is caused by mutations in the X-linked dystrophin (DMD) gene. The absence of dystrophin protein leads to progressive muscle weakness and wasting, disability and death. Existing animal models have been instrumental to understand the pathophysiology of DMD, but have limitations related to the type of mutation, the clinical phenotype, and the predictive value for molecular therapies. To establish a tailored large animal model of DMD, we deleted DMD exon 52 in male pig cells by gene targeting and generated offspring by nuclear transfer. DMD pigs exhibit absence of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive dystrophic changes of skeletal muscles, impaired mobility, muscle weakness, and a maximum life span of 3 months due to respiratory impairment. These findings render the DMD mutant pig a promising model for testing targeted genetic treatment approaches, such as exon skipping which partially restored in-frame dystrophin gene expression in our model. Regarding recent disappointing results of exon skipping in human DMD clinical trials, a valid animal model will be warranted. [ABSTRACT FROM AUTHOR]