Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the ß-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.

Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs.

BACKGROUND: Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. METHODS: First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. RESULTS: Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. CONCLUSIONS: Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.

Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology.

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.