Abstract
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.
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Acknowledgements
We thank Dr. Sangchul Kang and staff in the laboratory in Optipharm Corporation for histological analysis. This study was financially supported by grants from Korean Small and Medium Business Administration (SMBA, #S2316056) and Ministry of Agriculture, Food and Rural Affairs (MAFRA, 116085-3).
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Each author’s specific contributions to the work are indicated below. BC: research design, performed experiments (Targeting vector design and construction), data analysis, and writing the paper. SJK: performed of experiments (SCNT, pig generation), and data analysis. E-JL: performed experiments (Donor cell preparation and genotyping). SMA: performed experiments (Phenotypic analysis). JSL: performed experiments (SCNT and pig generation). D-YJ: performed experiments (pig generation). KL: data analysis, and writing the paper. J-TK: research design, performed experiments (SCNT, pig generation), data analysis, and writing the paper.
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BC, SJK, EJL, SMA, JSL, DYJ, and JTK are employees of Mgenplus Co., Ltd., a company that specializes in transgenic pig production.
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Fig. 1
Cell sorting using FACSAria™ cell sorter and T7E1 assay. A) GFP positive cells were sorted by the FACSAria™ cell sorter to enrich genetically engineered cells from fibroblasts transfected with INS specific CRISPR/Cas9 system. B) T7E1 assay was performed to identify fibroblasts carrying modified INS. (TIFF 418 kb)
Fig. 2
T7E1 assay in each single-cell derived colonies. Single-cell derived colonies were picked and cultured. Genomic DNA was harvested from picked colonies and used as template for PCR amplification and T7E1 assay to identify cell colonies with mutated INS. (TIFF 1282 kb)
Fig. 3
Schematic diagram of proinsulin and the alignment of deduced amino acid sequences. A) The alignment of deduced amino acid sequences between the wildtype and modified allele of sgRNA1-#33 donor cell. B) Schematic diagram of insulin and C-peptide expression from proinsulin and mutated proinsulin. Red arrowhead indicates cleavage site and black line shows disulfide bond. (TIFF 480 kb)
Fig. 4
Off-target effect of each sgRNA. PCR and T7E1 assay of off-target candidate of sgRNA1 (A; Accession No. 001244772) and sgRNA2 (B; Accession No. 001243805) was performed. (TIFF 626 kb)
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Cho, B., Kim, S.J., Lee, EJ. et al. Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system. Transgenic Res 27, 289–300 (2018). https://doi.org/10.1007/s11248-018-0074-1
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DOI: https://doi.org/10.1007/s11248-018-0074-1