Control of autoimmune diabetes in NOD mice by GAD expression or suppression in β cells

Science

Volumn 284, Issue 5417, 1999, Pages 1183-1187

  Yoon, Ji Won ^a,b,c   Yoon, Chang Soon ^a   Lim, Hye Won ^a   Huang, Qi Quan ^a   Kang, Yup ^b   Pyun, Kwang Ho ^d   Hirasawa, Kensuke ^a   Sherwin, Robert S ^c   Jun, Hee Sook ^a  

^a UNIVERSITY OF CALGARY   (Canada)

^b Ajou University   (South Korea)

^c YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^d Korea Research Institute of Bioscience and Biotechnology (KRIBB)   (South Korea)

Author keywords

[No Author keywords available]

Indexed keywords

AUTOANTIGEN; GLUTAMATE DECARBOXYLASE;

ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL MODEL; ARTICLE; AUTOIMMUNE DISEASE; GENE REPRESSION; INSULIN DEPENDENT DIABETES MELLITUS; MOUSE; NONHUMAN; PANCREAS ISLET BETA CELL; PATHOGENESIS; PRIORITY JOURNAL; PROTEIN EXPRESSION; TRANSGENIC MOUSE;

ADOPTIVE TRANSFER; ANIMALS; AUTOANTIGENS; AUTOIMMUNITY; DIABETES MELLITUS, TYPE 1; DNA, ANTISENSE; FEMALE; GENE EXPRESSION; GLUTAMATE DECARBOXYLASE; INSULIN; ISLETS OF LANGERHANS; ISLETS OF LANGERHANS TRANSPLANTATION; LYMPHOCYTE ACTIVATION; MALE; MICE; MICE, INBRED NOD; MICE, SCID; MICE, TRANSGENIC; T-LYMPHOCYTES; TRANSGENES;

ANIMALIA; MUS MUSCULUS;

EID: 0033553645     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5417.1183     Document Type: Article

References (28)

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10. S. Baekkeskov et al., Nature 347, 151 (1990); S. Baekkeskov et al., J. Clin. Invest. 79, 926 (1987); M. A. Atkinson, N. K. Maclaren, D. Scharp, P. E. Lacy, W. J. Riley, Lancet 335, 1357 (1990); M. A. Atkinson et al., ibid. 339, 458 (1992); J. Endl et al., J. Clin. Invest. 99, 2405 (1997).
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12. R. Tisch et al., ibid., p. 72.
13. The 7.6-kb RIP-AS-GAD65.67 full-length transgene was microinjected into fertilized eggs of (C57BL/6 × SJL)F2 mice [J. W. Gordon, Methods Enzymol. 225, 747 (1993)] ). The founder mice were screened for the incorporation of the transgene into the genornic DNA by Southern blotting and polymerase chain reaction (PCR). Six selected transgene-positive founder mice were then backcrossed with NOD mice for seven generations to establish AS-GAD65/67 transgenic NOD mice (AS-GAD-NOD). The mice were screened for the transmission of the transgene with PCR, with rGAD67-specific primers. Female mice were used to determine the incidence of diabetes unless specifically mentioned otherwise. Immunoreactive insulin content in the pancreas and plasma insulin concentration (seven mice per group) were determined, as previously described [J. W. Yoon, M. A. Lesniak, R. Fussganger, A. L. Notkins, Nature 264, 178 (1976)].
14. The 7.6-kb RIP-AS-GAD65.67 full-length transgene was microinjected into fertilized eggs of (C57BL/6 × SJL)F2 mice [J. W. Gordon, Methods Enzymol. 225, 747 (1993)] ). The founder mice were screened for the incorporation of the transgene into the genornic DNA by Southern blotting and polymerase chain reaction (PCR). Six selected transgene-positive founder mice were then backcrossed with NOD mice for seven generations to establish AS-GAD65/67 transgenic NOD mice (AS-GAD-NOD). The mice were screened for the transmission of the transgene with PCR, with rGAD67-specific primers. Female mice were used to determine the incidence of diabetes unless specifically mentioned otherwise. Immunoreactive insulin content in the pancreas and plasma insulin concentration (seven mice per group) were determined, as previously described [J. W. Yoon, M. A. Lesniak, R. Fussganger, A. L. Notkins, Nature 264, 178 (1976)].
15. A recombinant RIP-DIPA/pXF3 plasmid vector [D. Hanahan, Nature 315, 115 (1985)], which carries the RIP sequence [695 base pairs (bp)], 770 bp of diphtheria toxin gene (DIPA), the simian virus 40 (SV40) early gene terminator (400 bp) with a small t intron, and a polyadenylation site (I/A), was partially digested with Bam HI. The RIP-DIPA-I/A fragment (1.9 kb) isolated from the RIP-DIPA/pXF3 vector was cloned into the Xba I-Hind III site of the pBluescriptIISK vector (Stratagene). After removing the DIPA gene, we inserted rGAD65 cDNA (2.3 kb) (RIP-AS-GAD65) or rGAD67 cDNA (3.1 kb) (RIP-AS-GAD67) into the vector in antisense orientation. To construct the RIP- antisense GAD65.67 transgene, we ligated antisense GAD65 complete transcription unit, which had been isolated from RIP-AS-GAD65 by digestion with Sal I and Not I, at the Sal I site of RIP-AS-GAD67 to produce RIP-AS-GAD65.67. The 7.6-kb RIP-AS- GAD65.67 full-length transgene (Fig. 1A) was separated from pBluescriptIISK by digestion with Xho I and Not I.
16. k-AS-GAD-NOD mice and their respective transgene-negative littermates were prepared, and 20 μg of protein was separated by 10% SDS- polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane (Amersham), the membrane was reacted with a 1:3000 dilution of polyclonal rabbit antibody to GAD67 (Chemicon), and the GAD protein was detected by the biotin-streptavidin- peroxidase method, with a chemoluminescence system. As an internal control, the same membrane was probed with antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon).
17. Paraffin blocks were prepared and sectioned [P. Gilon, M. Tappaz, C. Remacle, Histochemistry 96, 355 (1991)], the sections were reacted with GAD1 monoclonal antibody (ATCC), and GAD expression was detected by the avidin-biotin-peroxidase complex method with a Vectastain Elite ABC kit (Vector Laboratories). Serial sections of the pancreas were also stained with guinea pig polyclonal antibody to insulin (Vector).
18. Y. Kang, K. S. Kim, K. H. Kim, J. W. Yoon, unpublished data.
19. The data are available at www.sciencemag.org/ feature/data/986073.shl
20. 7 cells per mouse) from acutely diabetic NOD mice intravenously into 6-week-old, irradiated, male H- AS-GAD-NOD mice and age-matched transgene- negative control male NOD mice. The animals were monitored as described above.
21. 7 cells per mouse) from acutely diabetic NOD mice intravenously into 6-week-old, irradiated, male H- AS-GAD-NOD mice and age-matched transgene- negative control male NOD mice. The animals were monitored as described above.
22. 7 cells per mouse) from acutely diabetic NOD mice intravenously into 6-week-old, irradiated, male H- AS-GAD-NOD mice and age-matched transgene- negative control male NOD mice. The animals were monitored as described above.
23. 3H]thymidine was measured.
24. k-AS-GAD-NOD mice or age- and sex-matched transgene-negative littermates and transplanted (400 islets per mouse) into the renal subcapsular region of acutely diabetic NOD mice [T. Utsugi, M. Nagata, T. Kawamura, J. W. Yoon, Transplantation 57, 1799 (1994)]. The animals were monitored for glycosuria and hyperglycemia as described above. Sections of the kidney capsules containing the transplanted islets were stained with haematoxylin and eosin and examined for lymphocytic infiltration.
25. J. F. Elliott et al., Diabetes 43, 1494 (1994).
26. D. Zekzer et al., J. Clin. Invest. 101, 68 (1998).
27. The total RNA was isolated from the pancreas of H-, M-, and L-AS-GAD-NOD mice and transgene-negative littermates with Trizol (Gibco BRL). The total RNA (20 μg) was separated by agarose-formaldehyde gel electrophoresis, transferred to a nylon membrane, and probed with in vitro transcribed sense rGAD65 or rGAD67 RNA. As an internal control, the same membrane was probed with antisense GAPDH RNA.
28. We thank D. Hanahan for the plasmid RIP-DIPA/pXF3 containing the RIP, A. Tobin for the rat GAD65 and rat GAD67 cDNA, K. Clarke and A. Kyle for editorial assistance, and B. Pinder for artwork. Supported by grants from the Medical Research Council of Canada, the Juvenile Diabetes Foundation International, the NIH (DK 45735 and DK 53015-01), the Alberta Heritage Foundation for Medical Research, and Korea Green Cross.