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Glutamic acid decarboxylase (GAD) is a pancreatic b cell autoantigen in humans and nonobese diabetic (NOD) mice. b Cell-specific suppression of GAD ex-pression in two lines of antisense GAD transgenic NOD mice prevented auto-immune diabetes, whereas persistent GAD expression in the b cells in the other four lines of antisense GAD transgenic NOD mice resulted in diabetes, similar to that seen in transgene-negative NOD mice. Complete suppression of b cell GAD expression blocked the generation of diabetogenic T cells and protected islet grafts from autoimmune injury. Thus, b cell-specific GAD expression isrequired for the development of autoimmune diabetes in NOD mice, and modulation of GAD might, therefore, have therapeutic value in type 1 diabetes.



Type 1

diabetes, or insulin-dependent diabe-tes mellitus, is the consequence of progres-siveTcell-mediated autoimmune destruction of pancreatic b cells (1, 2). However, the initial events that trigger the destruction of b cells are incompletely understood. Several b cell autoantigens have been implicated in the triggering of b cell-specific autoimmunity (1, 3). GAD is the strongest candidate in both humans and the NOD mouse, which is con-sidered the best animal model of the human disease (3-5). In NOD mice, GAD, as com-pared with other b cell autoantigens exam-ined, provokes the earliest T cell proliferative response (4, 5).

However, no unequivocal evidence exists to indicate that the b cell expression of GAD is required for the initia-tion of diabetes in NOD mice. To address this issue, we examined the effect of ively suppressing GAD expression in the b cells of diabetes-prone NOD mice. The suppression of b cell GAD expres-sion was achieved by producing transgenic

NOD mice with an antisense GAD transgene (6) for both isoforms of rat GAD cDNA (rGAD65 and rGAD67) under the control of the rat insulin promoter (RIP) (7) (Fig. 1A). Six lines of antisense GAD65.67 transgenic NOD mice were established, defined by rel-ative amount of transgene expression (Fig. 1,B and C).

The first three lines of transgenic mice with high, medium, and low levels of expression of antisense GAD65.67 in the b cells were designated H-AS-GAD-NOD, M-AS- GAD-NOD, and L-AS-GAD-NOD, re-spectively (Fig. 1, C and D), and the second three lines of antisense GAD65.67 transgenic mice were designated H k -AS-GAD-NOD, M k -AS-GAD-NOD, and L k -AS-GAD-NOD, respectively (Fig. 1, B and C). Protein immu-noblot analysis (8) revealed the complete suppression of b cell GAD expression in islets of H-AS-GAD-NOD mice, whereas

moderate and low suppression was found in M- and L-AS-GAD-NOD transgenic mice, respectively (Fig. 1E). In contrast, GAD ex-pression was detected equally in the brain tissue of transgene-negative NOD mice and the three lines of AS-GAD-NOD mice (Fig.1E). The b cell-specific suppression of GAD expression was confirmed in H-AS-GAD-NOD mice, whereas different amounts of GAD expression were seen in transgene-neg-ative, L-AS-GAD-NOD, and M-AS-GAD-NOD mice by immunohistochemical staining with antibodies to GAD and insulin (Fig. 1F) (9).

The three lines of AS-GAD-NOD mice were indistinguishable from the transgene-negative littermates in pancreatic insulin con-tent [H, 446 6 46; M, 461 6 51; L, 458 647; control, 451 6 42 (SD) (micrograms of insulin per gram of pancreas)] and plasmainsulin concentrations [H, 4.1 6 0.21; M, 4.3 6 0.19; L, 4.6 6 0.22; control, 4.5 6 0.18 (SD) (nanograms of insulin per milliliter of plasma)]. Similar results regarding the sup-pression of GAD expression in b cells and insulin content in pancreas and plasma were obtained in the second three lines of antisense GAD65.67 transgenic NOD mice.

To determine whether GAD expression in the b cells was required for the development of autoimmune diabetes in NOD mice, we monitored disease development in the three lines of AS-GAD-NOD mice and in trans-gene-negative littermates. None (0 of 15) of the H-AS-GAD-NOD mice developed diabe-tes by 40 weeks of age. In contrast, 67% (12 of 18) of the M-AS-GAD-NOD mice, 75% (12 of 16) of the L-AS-GAD-NOD mice, and 81% (17 of 21) of the transgene-negative littermates developed diabetes by the sameage (Fig. 2A). We examined the islet histol-ogy of the above groups at 20 weeks of age.

Over 80% of the examined H-AS-GAD-NOD islets were intact (Fig. 2, B and E), and less than 20% of the islets showed periinsulitis (Fig. 2B). In contrast, most of the M-AS-GAD-NOD and L-AS-GAD-NOD islets ex-amined showed moderate to severe insulitis, as did transgene-negative littermates (Fig. 2, B and F). In the second three lines of AS-GAD-NOD mice, diabetes appeared in 2.8% (1 of 36) of the H k -AS-GAD-NOD mice, 83.3% (15 of 18) of the M k -AS-GAD-NOD mice, and 80.8% (21 of 26) of the L k -AS-GAD-NOD mice by 40 weeks of age, where-as 85.7% (18 of 21) of the transgene-negative littermates developed diabetes (Fig. 2C). One animal in the H k -AS-GAD-NOD group did develop diabetes; whether this small differ-ence between the H-AS-GAD-NOD and H k -AS-GAD-NOD groups is due to leakiness in the H k -AS-GAD-NOD group or differences in the susceptibility genes is uncertain.

We also examined the islet histology of the H k -,M k -, and L k -AS-GAD-NOD mice at 19 to 20 weeks of age (Fig. 2D) and found no signif-icant difference in the extent of insulitis from that in the H-, M-, and L-AS-GAD-NOD groups (Fig. 2B).Our findings indicate that b cell GAD expression is a requirement for the develop-ment of diabetes in NOD mice. The complete prevention of diabetes in H-AS-GAD-NOD mice and the near complete prevention of diabetes in H k -AS-GAD-NOD mice are notlikely to be due to a nonspecific effect of the antisense transgene incorporated into the chromosomal DNA, because low or moderate suppression of GAD expression in M-, M k -,L-, and L k -AS-GAD-NOD mice carrying the same antisense transgene did not result in the prevention of diabetes (Fig. 2, A and C). To examine this issue further, we developed an-other control for the antisense GAD trans-genic NOD mice, namely, antisense trans-genic NOD mice carrying the antisense en-dogenous murine leukemia proviral env re-gion DNA under the control of the RIP.

Endogenous retroviral env protein, a putative b cell autoantigen, is expressed in the b cells of NOD mice (10). These antisense trans-genic NOD mice, unlike their GAD-sup-pressed counterparts, developed diabetes (79%, 15 of 19), as did the transgene-nega-tive littermates (82%, 9 of 11) (11), even though the antisense transgene was highly expressed and effectively blocked the endog-enous synthesis of viral protein. These results support the view that the prevention of dia-betes in antisense GAD transgenic NOD mice is not due to the nonspecific effect of an antisense transgene incorporated into chro-mosomal DNA.

To determine whether b cell-specific sup-pression of GAD expression specifically af-fects b cell-specific autoimmunity, we exam-ined the salivary gland, which also shows lymphocytic infiltration in diabetes-prone NOD mice. In contrast to the b cell, lympho-cytic infiltration in the salivary gland of H-AS-GAD-NOD mice was not prevented (Fig.2G), and sialitis was similar to that of trans-gene-negative littermates (Fig. 2H), indicat-ing that autoimmunity was not affected in other tissues.

We next examined whether the suppres-sion of GAD expression in the b cells inhibits disease development by blocking the gener-ation of b cell-specific diabetogenic T cells.

Splenocytes from 20-week-old nondiabetic female H-AS-GAD-NOD mice and age-matched nondiabetic transgene-negative lit-termates were transfused into 6- to 8-week-old NOD쯇evere combined deficiency dis-ease (NOD.scid) mice (12). None of the NOD.scid recipients (0 of 8) of splenocytes from H-AS-GAD-NOD mice developed dia-betes by 10 weeks after the transfer of spleno-cytes, whereas 90% (9 of 10) of the NOD. scid recipients of splenocytes from transgene-negative NOD mice developed diabetes with-in 9 weeks after transfer (Fig. 3A), as did NOD.scid recipients of splenocytes from acutely diabetic NOD mice. Similar results were obtained when we used splenocytes from H k -AS-GAD-NOD mice. Thus, the gen-eration of T cells capable of adoptively trans-ferring diabetes is blocked in the absence of GAD expression in the b cells. In addition, we determined which T cell subsets (CD4 1and CD8 1 ) are affected in H-AS-GAD-NOD mice. We found that the generation of both diabetogenic CD4 1 and CD8 1 T cells was blocked in the absence of GAD expression in the b cells (11).

Intravenous or intrathymus immunization of NOD mice with GAD suppresses T cell responses to GAD, heat shock protein (HSP) 60, carboxypeptidase H, and peripherin (4,5). To determine whether other b cell autoan-tigen-specific T cells developed in the ab-sence of GAD in the b cells, we examined the proliferative response of splenocytes from 8-(Fig. 3B), 12- (Fig. 3C), and 15- (Fig. 3D) week-old H-AS-GAD-NOD mice, transgene-negative littermates, and control NOD mice to GAD and other b cell autoantigens (HSP60 and insulin) (13). In contrast to the transgene-negative control group, no prolif-erative response to GAD was detected in H-AS-GAD-NOD mice at any age tested. T cells only from the latter transgenic mice at 15 weeks of age showed a small but insignif-icant proliferative response to HSP60 or in-sulin (Fig. 3, C and D). Similar results were obtained when we used splenocytes from H k -AS-GAD-NOD mice.

Thus, b cell-specific suppression of GAD gene expression dimin-ishes the T cell immune response to other b cell autoantigens as well as GAD. The susceptibility of GAD-suppressed b cells to attack by diabetogenic T cells derived from acutely diabetic NOD mice was evalu-ated by transplanting GAD-suppressed islets from H-AS-GAD-NOD mice or GAD-ex-pressing islets from young, transgene-nega-tive male NOD mice into the renal subcapsu-larregion of acutely diabetic NOD mice (14).

All recipients (6 of 6) of GAD-expressing islets showed a recurrence of diabetes (Fig.4A); most of the grafted islets showed mas-sive infiltration by mononuclear cells within 1 week and were destroyed within 2 weeks (Fig. 4, B and C, bottom). In contrast, none of the recipients (0 of 7) of GAD-suppressed islets showed a recurrence of diabetes up to 40 days after transplantation, at the termina-tion of the experiment (Fig. 4A). Further-more, over 80% of the grafted GAD-sup-pressed islets remained intact, and about 20% showed periinsulitis (Fig. 4, B and C, top). Most of these islets were positively stained by antibody to insulin. When we transplanted GAD-suppressed islets from H k -AS-GAD-NOD mice into the renal subcapsular region of acutely diabetic NOD mice, similar results were observed. In contrast, the transplanta-tion of env-suppressed islets from antisense env transgenic NOD mice resulted in their destruction within 2 weeks (11).

Thus, the env-suppressed islets were not resistant to the cytotoxic effect of diabetogenic T cells, sug-gesting that the resistance of GAD transgenic NOD islets is a specific rather than a nonspe-cific effect. In keeping with these results, when splenocytes from acutely diabetic NOD mice were transfused into 6-week-old, irradi-ated, male H-AS-GAD-NOD mice and age-and sex-matched transgene-negative litter-mates, none of the H-AS-GAD-NOD mice (0 of 9) developed diabetes, whereas 71% (5 of 7) of the transgene-negative control recipi-ents developed diabetes within 4 weeks after transfer (Fig. 4D), again demonstrating that GAD expression is required for autoimmune destruction of b cells.

Thus, GAD expression is essential for the induction of diabetogenic T cells, and diabetogenic T cells cannot provoke diabetes in NOD micein the absence of GAD from b cells.


Fig. 1. Structure of RIP-antisense GAD65.67 transgene, pedigree of antisense GAD trans-genic mouse lines, the expression of antisense GAD mRNA, and the suppression of GAD ex-pression in b cells of AS-GAD transgenic NOD mice.

(A) Diagram of RIP-antisense GAD65.67 transgene structure: RIP, SV40 small tintron, and a
polyadenylation site (I/A).

(B) Expression of an-tisense GAD tran by reverse tranase PCR. Two micrograms of total RNA was con-verted to cDNA with sense rat GAD67 prim-er (59-ATGACGTC-TCCTACGATACA-39),and the cDNA was am-plified with sense and antisense GAD67 prim-ers (59-CCCCTTGAGG-CTGGTAACCA-39). As an internal standard, hypoxanthine

guaninephosphoribosyl-trans-ferase mRNA was am-plified with the follow-ing primers: sense, 59-GTAATGATCAGTCAA-CGGGGGAC-39; and antisense, 59-CCAGCAAGCTTGCAACCTTAACCA-39. Lane M, 100-bp ladder. Each lane corresponds to the ear tag number shown in (C). Numbers at right are the size of amplified product in base pairs.

(C) Pedigree of AS-GAD transgenic mice. Eleven positive founder mice were obtained; six mice were ed on the basis of the expression of the antisense tran and backcrossed with NOD mice. Ear tag number: TN, transgene-negative littermates; C5, C57BL/6 mice; and NO, nontransgenic NOD mice. Transgene expression: H, high; M, intermediate; L, low; and 2, no expression of transgene.

(D) Northern (RNA) blot analysis (17) of antisense GAD trans in the first three different
lines of transgenic NOD mice (H-, M-, and L-AS-GAD-NOD) and transgene-negative littermates (-).

(E) Protein immunoblot analysis (8) of the suppression of GAD expression in pancreatic islets and brain tissue in the first three different lines of transgenic NOD mice (H-, M-, and L-AS-GAD-NOD) and transgene-negative littermates (-).

(F) Immunohistochemical staining (9) of pancreatic islets from 10-week-old H-, M-, and L-AS-GAD-NOD mice and transgene-negative [Tg (-)] littermates. Serial islet sections were stained with either hematoxylin and eosin (HE), antibody to GAD, or antibody to insulin. Magnification, x200.





Fig. 2. The effect of b cell-specific suppres-sion of GAD expression on the development of diabetes and insulitis.

(A) The incidence of di-abetes in the first three different lines of AS-GAD-NOD mice, at the
seventh generation with a NOD back-ground. Cumulative in-cidence of diabetes was determined by positive glycosuria and con-firmed by hyperglyce- mia (nonfasting blood glucose .16.7 mM) on 2 consecutive days up to 40 weeks of age.

(B) Histological exam-ination of insulitis in H-, M- and L-AS-GAD-NOD mice and trans-gene-negative litter-mates. Histological ex-amination of pancreat-ic islets at 20 weeks of age; shown are re-sults from five random-ly ed nondiabetic mice at 20 weeks of age (at least 20 islets per mouse examined). Grade: 0, normal islets;1, mononuclear infiltra-tion, largely in the pe-riphery, in less than 25% of the islet; 2, 25 to 50% of islet showing mononuclear infiltration; 3, over 50% of islet showing mononuclear infiltration; and 4, small, retracted islet with few mononuclear cells.

(C) Incidence of diabetes in the second three different lines of AS-GAD-NOD mice at the seventh generation with a NOD background.

(D) Histological examination of insulitis in H k -, M k -, and Lk -AS-GAD-NOD mice and transgene-negative littermates.
(E to H) Photomi- crographs of representative pancreatic islet (E and F) and salivary gland (G and H) sections from H-AS-GAD-NOD mice and transgene-negative NOD littermates. Paraffin sections of pancreas or salivary gland were stained by HE.

(E) H-AS-GAD-NOD pancreatic section (intact islets).

(F) Transgene-negative NOD littermate pancreatic section (severe lymphocytic infiltration).

(G) Salivary gland sections of H-AS-GAD-NOD mice (severe lymphocytic infiltration).

(H) Salivary gland sections of transgene-negative NOD littermate (severe lymphocytic infiltration).
Magnification, x400.

Fig. 3. The effect of b cell-specific suppression of GAD expression on the development of b cell-cytotoxic T cells and T cell immune responses to islet autoantigens. (A) Incidence of diabetes in 6-to 8-week-old female NOD.scid mice that received splenocytes (1 3 10 7 cells per mouse) isolated from 20-week-old H-AS-GAD-NOD mice (n 5 8), age-matched transgene-negative littermates (n 5 10), or newly diabetic NOD mice (n 5 9). (B to D) Splenic T cell proliferative response to islet antigens.

Splenocytes isolated from 8-week-old (B), 12-week-old (C), and 15-week-old (D) female H-AS-GAD-NOD mice, female transgene-negative littermates, or female NOD mice were reacted with GAD peptide, recombinant human GAD65 protein, HSP60, porcine insulin, or ovalbumin, and the cells were incubated with 1 mCi of [ 3 H]thymidine. Proliferation was determined by [ 3 H]thymidine uptake. Data are expressed as stimulation indices (SI) 6 SD of the mean from five individual mice, tested in triplicate. Cutoff value of SI was 2.0. <, P , 0.01; <, P , 0.05 as compared with transgene-negative littermates.

Fig. 4. Protection of GAD-suppressed b cells from autoim-mune attack by diabe-togenic T cells. (A) Prevention of the re-currence of diabetes by the transplantation of GAD-suppressed is-lets into the subrenal capsule of acutely di-abetic NOD mice.

Acutely diabetic NOD mice received islets (400 islets per mouse) from 4-week-old male H-AS-GAD-NOD (n 57) or age- and sex-matched transgene-negative littermates (n 5 6). Blood glucose was measured every other day after islet transplantation. (B) In-sulitis grade in islet grafts from H-AS-GAD-NOD mice and trans-gene-negative litter-mates.

The insulitisgrades are described in Fig. 2B. (C) Photomicrographs of representative islet grafts from H-AS-GAD-NOD mice (top) (intact islets in the kidney capsule) and transgene-negative littermates (bottom) (massive infiltration of islets by mononuclear cells in the kidney capsule). Magnification,x400. (D) Adoptive transfer of diabetes to H-AS-GAD-NOD mice or transgene-negative littermates by acutely diabetic splenocytes. Irradiated,6-week-old male H-AS-GAD-NOD mice (n 5 9) or transgene-negative littermates (n 5 7) received splenocytes (1 3 10 7 cells per mouse) from acutely diabetic NOD mice.

R E P O R T S
14 MAY 1999 VOL 284 SCIENCE www.sciencemag.org 1186


References and Notes
1. R. Tisch and H. McDevitt, Cell 85, 291 (1996); A. A.Rossini, D. L. Greiner, H. P. Friedman, J. P. Mordes, Diabetes Rev. 1, 43 (1993); F. S. Wong and C. A. Janeway Jr., Res. Immunol. 148, 327 (1997); J. W. Yoon, H. S. Jun, P. S. Santamaria, Autoimmunity 27, 109 (1998).

2. M. Nagata and J. W. Yoon, Diabetes 41, 998 (1992).

3. 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).

4. D. L. Kaufman et al., Nature 366, 69 (1993).

5. R. Tisch et al., ibid., p. 72.

6. The 7.6-kb RIP-AS-GAD65.67 full-length transgene was microinjected into fertilized eggs of (C57BL/6 3 SJL)F2 mice [ J. W. Gordon, Methods Enzymol. 225,747 (1993)]). The founder mice were screened for the incorporation of the transgene into the genomic DNA by Southern blotting and polymerase chain reaction (PCR). Six ed 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 specifical- ly mentioned otherwise. Immunoreactive insulin con-tent in the pancreas and plasma insulin concentration (seven mice per group) were determined, as previ-ously described [ J. W. Yoon, M. A. Lesniak, R. Fuss- ganger, A. L. Notkins, Nature 264, 178 (1976)].

7. 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 diph-theria 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 digest-ed 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 pBlueIISK 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 tranion 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 sepa- rated from pBlueIISK by digestion with Xho I and Not I.

8. The total protein extracts from the islets or brain tissue of H-, M-, and L-AS-GAD-NOD mice as well as H k -, M k - and L k -AS-GAD-NOD mice and their respec- tive transgene-negative littermates were prepared,and 20 mg of protein was separated by 10% SDS- polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane (Amersham), the mem- brane was reacted with a 1: 3000 dilution of poly- clonal rabbit antibody to GAD67 (Chemicon), and the GAD protein was detected by the biotin-streptavidin- peroxidase method, with a chemoluminescence sys- tem. As an internal control, the same membrane was probed with antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon).

9. Paraffin blocks were prepared and sectioned [P. Gilon,M. Tappaz, C. Remacle, Histochemistry 96, 355 (1991)], the sections were reacted with GAD1 mono-clonal antibody (ATCC), and GAD expression was detected by the avidin-biotin-peroxidase complex method with a Vectastain Elite ABC kit (Vector Lab-oratories). Serial sections of the pancreas were also stained with guinea pig polyclonal antibody to insulin (Vector).

10. Y. Kang, K. S. Kim, K. H. Kim, J. W. Yoon, unpublished data.

11. The data are available at www.sciencemag.org/feature/data/986073.shl

12. To measure the generation of diabetogenic T cells in GAD-suppressed transgenic NOD mice, we did adop-tive transfer [T. Kawamura, M. Nagata, T. Utsugi, J. W.Yoon, J. Immunol. 151, 4362 (1993); M. Nagata, P.Santamaria, T. Utsugi, J. W. Yoon, ibid. 152, 2042 (1994)]. Splenocytes isolated from 20-week-old non-diabetic female H-AS-GAD-NOD mice, H k -AS-GAD-NOD mice, and their respective age-matched, trans-gene-negative littermates were transfused intrave-nously (1 3 10 7 cells per mouse) into 6- to 8-week-old NOD.scid mice. The animals were monitored three times per week for glycosuria (.12) and hy-perglycemia (.16.7 mM) [ J. W. Yoon, M. M. Rod-rigues, C. Currier, A. Notkins, Nature 296, 566 (1982)]. To determine whether the GAD-suppressed b cells are protected from autoimmune attack by diabetogenic T cells, we transfused splenocytes (1 310 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.

13. Splenocytes were isolated from individual 8-, 12-,and 15-week-old H-AS-GAD-NOD mice or H k -AS-GAD-NOD mice, their respective transgene-negative littermates, and control NOD mice, and a prolifera-tion assay was performed as described previously (2,4, 5). The splenocytes (1 3 10 6 cells per well) were plated in 200 ml of culture medium in triplicate and reacted with GAD peptide (mixed 17, 34, and 35 peptides; 7 mM) (4), recombinant human GAD65 protein (Syntax), HSP60 (StressGen), porcine insulin (Sigma), or ovalbumin (Sigma) at 20 mg/ml for 72 hours and pulsed with 1 mCi of [ 3 H]thymidine. The incorporation of [ 3 H]thymidine was measured.

14. Islets were isolated from 4-week-old male H-AS-GAD-NOD or H 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, Trans-plantation 57, 1799 (1994)]. The animals were mon-itored 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 in?tration.

15. J. F. Elliott et al., Diabetes 43, 1494 (1994).

16. D. Zekzer et al., J. Clin. Invest. 101, 68 (1998).

17. The total RNA was isolated from the pancreas of H-,M-, and L-AS-GAD-NOD mice and transgene-nega-tive littermates with Trizol (Gibco BRL). The total RNA (20 mg) was separated by agarose-formalde- hyde gel electrophoresis, transferred to a nylon membrane, and probed with in vitro transcribed sense rGAD65 or rGAD67 RNA. As an internal con-trol, the same membrane was probed with antisense GAPDH RNA.

18. 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 Her- itage Foundation for Medical Research, and Korea Green Cross. 23 October 1998; accepted 25 March 1999

 

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