Original Article Genetic Reconstitution of Autoimmune Type 1 Diabetes With Two Major Susceptibility Genes in the Rat Norihide Yokoi,1,2 Chihiro Hayashi,1 Yuuka Fujiwara,1 He-Yao Wang,3 and Susumu Seino1 The Komeda diabetes-prone (KDP) rat is an animal model LETL, and KDP rats have the identical MHC haplotype, u u u u u of human autoimmune type 1 diabetes. We have previously RT1 (RT1.A B D C ), while LEW.1AR1-iddm rats have shown that two major susceptibility genes, the major his- a recombinant haplotype, RT1r2 (RT1.Aa Bu Du Cu). How- tocompatibility complex (MHC) RT1u haplotype and Cblb ever, the MHC class II haplotype (RT1.Bu Du)iscommon (Casitas B-lineage lymphoma b) mutation, are responsible to all of the rat models of type 1 diabetes, suggesting that for the development of diabetes in KDP rats, suggesting a the class IIu haplotype confers susceptibility to type 1 two-gene model for development of the disease. To confirm diabetes in rats (11). the two-gene model, we produced a congenic strain carry- To confirm the susceptibility genes in type 1 diabetes, ing mutated Cblb alleles of the KDP rat on a non-KDP genetic background harboring the RT1u haplotype on its several studies on the genetic reconstitution of the disease MHC. Despite the low incidence and delayed onset of on nondiabetic genetic backgrounds have been performed diabetes, the congenic strain did develop the disease, but have been unsuccessful to date (12–14). The congenic indicating that type 1 diabetes can be reconstituted on a rat strain carrying two major susceptibility alleles, the non-KDP genetic background with the RT1u haplotype and RT1u haplotype and Lyp/Ian4l1, of the BB rat on the F344 Cblb mutation. Similar to observations in KDP rats, the genetic background did not exhibit diabetes or insulitis congenic strain showed insulitis and thyroiditis, symptoms (12). Similarly, the congenic strain carrying the Lyp/Ian4l1 of autoimmunity. The low incidence and delayed onset of allele of the BB rat on the F344 background showed the the disease strongly suggest involvement of genetic modi- lymphopenia phenotype without insulitis and diabetes fiers; the congenic strain established in this study should be useful for the mapping and identification of such modi- (13). In addition, the congenic strains carrying susceptibil- fiers. Diabetes 56:506–512, 2007 ity alleles of the NOD mouse on the C57BL/6 (B6) genetic background did not develop diabetes, but insulitis was occasionally observed in some of the animals (14). In contrast, genetic reconstitution of another autoimmune disease, systemic lupus erythematosus, has successfully ype 1 diabetes is an autoimmune disease in been performed. In this model, the combination of three which both genetic and environmental factors susceptibility genes on the B6 genetic background resulted are involved. Only the major histocompatibility in development of the disease (15). Tcomplex (MHC) has been identified as a major Based on our previous studies (16,17), we proposed a genetic factor, but several other genes may be involved in two-gene model for the development of type 1 diabetes in INS the development of the disease, such as (insulin), KDP rats (18). In this model, two major susceptibility CTLA4, and PTPN22 (1,2). Several spontaneous animal genes, the RT1u haplotype and Cblb (Casitas B-lineage models of type 1 diabetes have been established, including lymphoma b) mutation determined tissue specificity to the nonobese diabetic (NOD) mouse (3), the BioBreeding pancreatic ␤-cells and autoimmune reaction, respectively. (BB) rat (4,5), the Long-Evans Tokushima Lean (LETL) rat However, two important questions remain to be answered. (6), the Komeda diabetes-prone (KDP) rat (7), and the First, can type 1 diabetes be reconstituted on non-KDP LEW.1AR1-iddm rat (8). Genetic analyses demonstrate genetic backgrounds with these two major susceptibility that MHC is the major genetic factor underlying the genes? And second, are other genetic factors involved in development of diabetes in all of these models. In addition, the development of this disease in KDP rats? To answer combinations of MHC and other susceptibility genes may these questions, we have produced and investigated a contribute to the development of the disease (9,10). BB, congenic strain carrying the mutated Cblb allele of the KDP rat on a non-KDP genetic background harboring the u From the 1Division of Cellular and Molecular Medicine, Kobe University RT1 haplotype on its MHC. Graduate School of Medicine, Chuo-ku, Kobe, Japan; the 2Clinical Genome Informatics Center, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan; and the 3Shanghai Institute of Materia Medica, Chinese Academy RESEARCH DESIGN AND METHODS of Sciences, Shanghai, China. Address correspondence and reprint requests to Susumu Seino, Division of Tester Moriyama (TM) rats were obtained from Dr. T. Serikawa at the Institute Cellular and Molecular Medicine, Kobe University Graduate School of Medi- of Laboratory Animals, Kyoto University. Both TM and KDP rats (19) were cine, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected]. maintained under specific pathogen-free (SPF) conditions with a 12-h light/ Received for publication 24 July 2006 and accepted in revised form 23 dark cycle. A commercial diet (CE-2; CLEA Japan, Tokyo, Japan) and water September 2006. were available ad libitum at the Institute for Experimental Animals, Kobe MHC, major histocompatibility complex; SPF, specific pathogen free. University School of Medicine. The TM and KDP rats were crossed to produce DOI: 10.2337/db06-1027 the F1 progeny. Following this, six successive backcrosses (N7) to the TM rats © 2007 by the American Diabetes Association. were performed. An intercross between the N7 animals was conducted to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance obtain the congenic strain (N7F1) carrying the mutated Cblb allele of the KDP with 18 U.S.C. Section 1734 solely to indicate this fact. rat on the TM genetic background. This congenic strain was maintained at the 506 DIABETES, VOL. 56, FEBRUARY 2007 N. YOKOI AND ASSOCIATES same facility. All animal experiments were approved by the Committee on developed the disease at ϳ120 days of age (156.3 Ϯ 9.4 Animal Experimentation, Kobe University School of Medicine (permission no. days, n ϭ 9). Despite the low incidence and the delay of P-031207) and carried out in accordance with the Guidelines for Animal Experimentation at Kobe University. The KDP rats are available from Japan onset, it is noteworthy that type 1 diabetes was reconsti- SLC (Shizuoka, Japan). The congenic strain (TM.KDP-Cblb) has been depos- tuted on the non-KDP genetic background with the two u ited (ref. no. 0354) and is available from the National Bio-Resource Project of major susceptibility genes (RT1 haplotype and Cblb Rat in Japan (www.anim.med.kyoto-u.ac.jp/nbr/). mutation). Phenotyping. Phenotyping was completed using a previously described We also compared body weight and changes in blood protocol (16). Diabetes was defined as glycosuria positivity and blood glucose glucose level in the congenic and KDP strains (Fig. 1B–D). levels Ն250 mg/dl under ad libitum dietary conditions. Data were obtained from five generations (N7F1 to N7F5) of the congenic strain and one At 210 days of age, both females and males in the congenic generation of the KDP strain. strain exhibited higher body weight than KDP rats (KDP Genotyping. Simple sequence-length polymorphism markers used in this vs. congenic: female 230 Ϯ 4.5 vs. 246.5 Ϯ 3.7 g, P ϭ 0.014; study have been described elsewhere (RATMAP, available at http://ratmap- male 377.8 Ϯ 5.7 vs. 450.5 Ϯ 10.2 g, P Ͻ 0.0001). In .gen.gu.se/; Rat Genome Database, available at http://rgd.mcw.edu/; 16,17,19). contrast, there was no difference in changes in blood Genotyping was performed as described in prior investigations (16,17,19). glucose levels between the strains, except for age of onset Histological analysis. Histological analysis was performed as described previously (7,16), with some modifications. Briefly, tissues were fixed in 10% of diabetes: blood glucose levels elevated rapidly from formalin, and paraffin sections were stained with hematoxylin and eosin. ϳ100 to ϳ400 mg/dl within a week in both the congenic Serial sections were viewed via light microscopy by an examiner blind to the and KDP strains. experimental conditions of the animals. Each animal was rated on the degree Degree of insulitis of the congenic and KDP strains. of insulitis, which ranged from none to severe (none [0%], slight [Ͼ0 and Յ5%], We then investigated the degree of insulitis shortly after mild [Ͼ5 and Յ20%], moderate [Ͼ20 and Յ70%], and severe [Ͼ70%]) based the onset of diabetes in diabetic animals and at 210 days of mainly on the percentage of moderately and severely infiltrated islets. The term “slight insulitis” refers to at least one infiltrated islet across the sections age in nondiabetic animals (Fig. 2). In the congenic strain, and a percentage of infiltrated islets of Յ5%. Animals were also rated on the the Cblb homozygous mutants (n ϭ 35) showed slight to degree of thyroiditis, which ranged from none to severe (none [0%], slight to severe insulitis, while heterozygous (n ϭ 22) and wild-type mild [Ͼ0% and Յ20%], and moderate to severe [Ͼ20%]) based on the (n ϭ 14) animals exhibited none or only slight insulitis percentage of infiltrated regions in thyroid sections. Data were obtained from (TM.KDP-Cblb homozygotes vs. heterozygotes, ␹2 [df ϭ 4] five generations (N7F1 to N7F5) of the congenic strain and one generation of ϭ 43.7, P Ͻ 0.0001; TM.KDP-Cblb homozygotes vs.