Kaichun Wei

Mapping and characterization of quantitative trait loci for non-insulin-dependent diabetes mellitus with an improved genetic map in the Otsuka Long-Evans Tokushima Fatty rat

Abstract. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. We have previously reported four quantitative trait loci (QTLs) responsible for NIDDM on Chromosomes (Chrs) 7, 14, 8, and 11 (Nidd1–4/of for Non-insulin-dependent diabetes1–4/oletf) by a whole-genome search in 160 F2 progenies obtained by mating the OLETF and the Fischer-344 (F344) rats. Our present investigation was designed to identify and characterize novel QTLs affecting NIDDM by performing a genome-wide linkage analysis of genes for glucose levels and body weight and analysis for gene-to-gene and gene-to-body-weight interactions on an improved genetic map with a set of 382 informative markers in the 160 F2 progenies. We have identified seven novel QTLs on rat Chrs 1 (Nidd5 and 6/of), 5 (Nidd7/of), 9 (Nidd8/of), 12 (Nidd9/of), 14 (Nidd10/of) and 16 (Nidd11/of) which, together with the Nidd1–4/of, account for a total of ∼60% and ∼75% of the genetic variance of the fasting and postprandial glucose levels, respectively, in the F2. While the OLETF allele corresponds with increased glucose levels as expected for the novel QTLs except Nidd8 and 9/of, the Nidd8 and 9/of exhibit heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. There are epistatic interactions between Nidd1 and 10/of and between Nidd2 and 8/of. Additionally, our results indicated that the Nidd6 and 11/of could also contribute to an increase of body weight, and that the other five QTLs could show no linkage with body weight, but Nidd8,9, and 10/of have an interaction with body weight.

Identification of novel non-insulin-dependent diabetes mellitus susceptibility loci in the Otsuka Long-Evans Tokushima Fatty rat by MQM-mapping method

Abstract. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. We have previously identified 11 quantitative trait loci (QTLs) responsible for NIDDM susceptibility on Chromosomes (Chrs) 1, 5, 7, 8, 9, 11, 12, 14, and 16 (Nidd1–11/of for Non-insulin-dependent diabetes1–11/oletf) by using the interval mapping method in 160 F2 progenies obtained by mating the OLETF and the Fischer-344 (F344) rats. MQM-mapping, which was applied for QTL analysis based on multiple-QTL models, is reported to be more powerful than interval mapping, because in the process of mapping one QTL the genetic background, which contains the other QTLs, is controlled. Application of MQM-mapping in the F2 intercrosses has led to a revelation of three novel QTLs on rat Chrs 5 (Nidd12/of), 7 (Nidd13/of), and 17 (Nidd14/of), in addition to Nidd1–11/of loci. The three QTLs, together with the Nidd1–11/of, account for a total of ∼70% and ∼85% of the genetic variance of the fasting and postprandial glucose levels, respectively, in the F2. While the OLETF allele corresponds with increased glucose levels as expected for Nidd12 and 14/of, the Nidd13/of exhibits heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. There is epistatic interaction between Nidd2 and 14/of. Additionally, our results indicated that the novel QTLs could show no linkage with body weight, but Nidd12/of has an interaction with body weight.

A major quantitative trait locus co-localizing with cholecystokinin type A receptor gene influences poor pancreatic proliferation in a spontaneously diabetogenic rat

Abstract. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for pancreatic proliferation, which may be the critical pathogenetic event in NIDDM development. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for poor pancreatic proliferation by examining compensatory proliferation of the pancreatic remnant after partial pancreatectomy and performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We identified a highly significant QTL on rat Chromosome 14 with a maximum lod score of 16.7, which accounts for 55% of the total variance. The QTL co-localizes with the gene encoding cholecystokinin type A receptor (CCKAR) which is likely to mediate the trophic effect of cholecystokinin on pancreas and is defective in the OLETF rat.

AN INTEGRATED GENETIC MAP OF THE RAT WITH 562 MARKERS FROM DIFFERENT SOURCES

Abstract. Genetic maps are the primary resources for genetic study. Genetic map construction was quite difficult in the past decade for lack of polymorphic markers. This situation has been changed since the development of microsatellite markers or simple sequence length polymorphisms (SSLPs) because they are abundant and more polymorphic. Here we report the construction of an integrated genetic map of the rat derived from two F2 intercrosses. A map of 376 markers from 160 (OLETF × F344)F2 progenies and a map of 333 markers from 71 (F344 × LEC)F2 animals are integrated by use of common set of 120 anchor markers chosen to be spaced at an average of 15 cM in the genome. The resulting integrated map with 194 newly developed rat markers from WIBR/MIT CGR, 269 Mit/Mgh markers, 94 Wox markers, and 5 markers of various origins covers the majority of 21 chromosomes of the rat with a total genetic distance of 1797 cM and an average marker spacing of 3.2 cM. The current map provides detailed information for markers from different sources and, therefore, should be helpful to the research community.

Chromosomal mapping of the T-helper immunodeficiency (thid) locus in LEC rats.

The mechanism underlying the development of thymocytes into CD4+8± or CD4±8+ cells is not fully understood. Mutant strains have served as an invaluable tool for the study of such a mechanism. Lymphopoietic stem cells from bone marrow migrate to the thymus where they undergo gene rearrangement of the T-cell receptor (TCR), and then express TCR together with their coreceptor molecules, CD4 and CD8, on the developing thymocytes. Coengagement of TCR and coreceptor CD4 or CD8 on the maturing cells by their thymic MHC molecules will determine their cell fate. CD4+8+ thymocytes that recognize major histocompatibility complex (MHC) class I molecules on the thymic epithelial cells differentiate into CD4±8+ cells, whereas CD4+8+ thymocytes that recognize MHC class II mature as CD4+8± cells. The molecular basis of such an influence is largely unknown (von Boehmer 1994, 1996; Fink and Bevan 1995). The Long-Evans Cinnamon (LEC) rat, as far as is known, was the first naturally occurring rat mutant with a specific defect in thymocyte development. The development of CD4+8+ thymocytes into CD4+8± cells was prevented in the thymus of the LEC rat, but the development of CD4+8+ thymocytes into CD4±8+ cells was normal (Agui et al. 1990). Thymocytes from the LEC rat contained less than 1% CD4+8± cells. Unexpectedly, a small number of CD4+ T cells were present in the peripheral lymphoid organs (Agui et al. 1990). These CD4+ T cells were shown not to function as helper T cells, since LEC rats did not produce antibodies against T-cell-dependent antigen, SRBC, but did produce antibodies against T-cell-independent antigen (DNPconjugated Ficoll) (Agui et al. 1990). Moreover, CD4+ T cells in LEC rats failed to secrete IL-2 upon Concanavalin A stimulation (Agui et al. 1990; Sakai et al. 1993). Such a defect in LEC rats was brought about by a single autosomal recessive gene designated as T-helper immunodeficiency (thid) (T. Yamada et al. 1991). The inability to generate CD4+8± thymocytes together with physical and functional deficiencies in CD4+ T cells suggests that the LEC rat may serve as a model for further studies in thymic selection and T-cell functions. Here we performed linkage mapping of the mutation. The linkage map surrounding thid on rat chromosome 1 can serve as the starting point for further characterization of the mutation9s molecular basis. LEC/Tj, LEA/Tj, WKAH/Tj, and F344/Tj inbred rats were maintained in our laboratory under specific pathogenfree conditions. BN/Kyo rats were kindly provided by J. Yamada, University of Kyoto, Japan. Four backcrosses between LEC and one of the normal rats were made. Thymocytes prepared from the progenies of the backcrosses at the age of 4±6 weeks were first stained with biotinylated anti-rat CD8 mAb (OX8) (Brideau et al. 1980), and then stained with PE-conjugated streptavidin and FITC-conjugated anti-rat CD4 mAb(W3/25) (Williams et al. 1977). Stained cells were analyzed with a FACScan and Consort 30 software program (Becton Dickinson, Mountain View, Calif.). Animals with a percentage of CD4+8± thymocytes less than 1% were scored as thid/thid homozygotes, while those greater than 5% were scored as thid/+ heterozygotes (Fig. 1). DNA was also extracted from the liver of the backcross progenies using phenol and chloroform (Sambrook et al. 1989). Most of the rat microsatellite primers (Jacob et al. 1995; Gauguier et al 1996) were purchased from Research Genetics, and the remaining primers were synthesized according to published sequences (Serikawa et al. 1992). The marker was typed by polymerase chain reaction (PCR) with DNA from the backcross progenies and the above primers. PCR reaction mixture (10 ml) contained 25 ng K. Wei ? T. Yamada ? K. Matsumoto ( ) Institute for Animal Experimentation, University of Tokushima School of Medicine, Kuramoto 3, Tokushima 770, Japan

A MUTANT STRAIN (LEC) OF RAT WITH LOW DEGREE OF ZINC-INDUCED HEPATIC METALLOTHIONEIN GENE EXPRESSION

A mutant strain (LEC) of rats was found to possess the feature of low degree of the zinc-induced hepatic metallothionein (MT) gene expression due to an alteration of the transcription factor concerned in the gene expression. Northern blot analyses showed that the amount of MT-1 mRNA induced by intraperitoneal zinc injection is smaller in LEC mutant rat liver than in normal rat liver, while the amount of MT-1 mRNA induced by copper injection is indistinguishable between LEC and normal rat livers. Gel retardation assays showed that LEC and normal rat livers are different in the nuclear protein which binds to the metal-responsive element (MRE) of the MT gene in a zinc-dependent manner, and that the efficiency of the zinc-dependent binding of the nuclear protein to the MRE is lower in LEC rat liver than in normal rat liver. LEC rat should provide a useful model to understand the transcription factor concerned in the MT gene expression by zinc.