Congenic Rat Strains Research Articles

Establishment of thymoma-prone congenic rat strain, ACI.BUF/Mna-Tsr1/Tsr1

To confirm the presence of the susceptible gene for the thymoma development in the region that was assumed by the previous linkage study by Oyabu et al. (J Natl Cancer Inst 91:279-282, 1999), we tried to establish a congenic strain of rats. Backcrossings between the BUF/Mna strain as a donor strain and the ACI/NMna strain as an inbred partner were repeated for 12 generations, examining whether rats had the thymoma development region, and then homozygous rats were yielded by mating among the heterozygotes. To detect the phenotypic expression, heterozygous ACI.BUF/Mna-Tsr1/+ (ACI-Tsr1/+) rats were generated by crossing female ACI.BUF/Mna-Tsr1/Tsr1 (ACI-Tsr1/Tsr1) rats with male ACI/NMna rats and were maintained for 24 months. These ACI-Tsr1/+ rats produced thymoma in 71%, showing a dominant trait. The thymomas were of the lymphocyte predominant type, as those developed in rats of the original BUF/Mna strain. Thus, a new rat congenic strain, ACI-Tsr1/Tsr1, was established, revealing that thymoma develops in the dominant trait in ACI-Tsr1/+ rats.

Characterization of the encephalitogenic immune response in a model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) can be actively induced with the extracellular domain of myelin oligodendrocyte glycoprotein (MOG 1-125). MOG-EAE closely mimics multiple sclerosis (MS) especially as far as demyelination, lesion formation and axonal pathology are concerned. MOG 91-108 is the encephalitogenic stretch within MOG 1-125 in two EAE-susceptible MHC congenic LEW rat strains [LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n))] and DA (RT1(av1)) rats. In LEW.1AV1 rats, disease could be induced with MOG 96-104 and to a lesser extent with MOG 98-106, whereas in LEW.1N rats, only MOG 98-106 was pathogenic. Both peptides bound well to their restricting MHC class II molecules, i.e., RT1.D(n) in the LEW.1N rat and RT1.B(a) in the LEW.1AV1 rat. TCR spectratyping of MOG 91-108 immunized LEW.1N, LEW.1AV1 and DA rats revealed that MHC class II determined the TCRBV preference of CNS infiltrating T cells. The data demonstrate that the most critical factor in inducing MS like pathology is presentation of autoantigenic peptides on MHC class II molecules resulting in demyelination and axonal pathology.

Physiogenomic strategies and resources to associate genes with rat models of heart, lung and blood disorders

As is the case for many human disorders, cardiovascular disease is a complex ailment exhibiting a multifactorial mode of transmission. Rat models have been developed to aid in the analysis of this complex genetic and phenotypic disorder. The purpose of this brief review is to describe current gene expression profiling strategies that have been implemented to search for candidate causative genes of disease phenotypes in animal models. Strategies include integrating gene expression information with linkage analysis, expression profiling chromosome-substituted and/or congenic rat strains, correlating gene expression with physiological data across a panel of rodent strains, and linking expression quantitative trait loci to physiological quantitative trait loci. A primary goal of these strategies is to narrow and prioritize the search for causal genes of physiological interest. Also discussed are ways to harness two recent publicly available resources that have been created to investigate the role of genes and environment on cardiovascular physiology and pathophysiology.

Identification of Candidate Genes for Alcohol Preference by Expression Profiling of Congenic Rat Strains

A highly significant quantitative trait locus (QTL) on chromosome 4 that influenced alcohol preference was identified by analyzing crosses between the iP and iNP rats. Congenic strains in which the iP chromosome 4 QTL interval was transferred to the iNP (NP.P) exhibited the expected increase in alcohol consumption compared with the iNP background strain. This study was undertaken to identify genes in the chromosome 4 QTL interval that might contribute to the differences in alcohol consumption between the alcohol-naïve congenic and background strains. RNA from 5 brain regions from each of 6 NP.P and 6 iNP rats was labeled and analyzed separately on an Affymetrix Rat Genome 230 2.0 microarray to look for both cis-regulated and trans-regulated genes. Expression levels were normalized using robust multi-chip average (RMA). Differential gene expression was validated using quantitative real-time polymerase chain reaction. Five individual brain regions (nucleus accumbens, frontal cortex, amygdala, hippocampus, and striatum) were analyzed to detect differential expression of genes within the introgressed QTL interval, as well as genes outside that region. To increase the power to detect differentially expressed genes, combined analyses (averaging data from the 5 discrete brain regions of each animal) were also carried out. Analyses within individual brain regions that focused on genes within the QTL interval detected differential expression in all 5 brain regions; a total of 35 genes were detected in at least 1 region, ranging from 6 genes in the nucleus accumbens to 22 in the frontal cortex. Analysis of the whole genome detected very few differentially expressed genes outside the QTL. Combined analysis across brain regions was more powerful. Analysis focused on the genes within the QTL interval confirmed 19 of the genes detected in individual regions and detected 15 additional genes. Whole genome analysis detected 1 differentially expressed gene outside the interval. Cis-regulated candidate genes for alcohol consumption were identified using microarray profiling of gene expression differences in congenic animals carrying a QTL for alcohol preference.

Neuronal Responsiveness to Central Na + in 2 Congenic Strains of Dahl Salt-Sensitive Rats

Dahl salt-sensitive rats show increased Na(+) entry into the brain on high salt intake and increased sympathetic and pressor responses to central Na(+). We examined C10QTL2 and C17QTL to test whether they contribute to these phenotypes. In Dahl salt-sensitive, Lewis, and C10S.L16, and C17S.L2 congenic rats on a high salt diet for 8 to 10 days, blood pressure and heart rate were higher in Dahl salt-sensitive versus others and in C10S.L16 and C17S.L2 versus Lewis rats. Cerebrospinal fluid [Na(+)] increased by approximately 5 mmol/L in Dahl salt-sensitive, C10S.L16, and C17S.L2 compared with Lewis rats. In rats on a regular salt diet, 8-minute intracerebroventricular infusions of artificial cerebrospinal fluid with increasing [Na(+)] caused increases in blood pressure, heart rate, and renal sympathetic nerve activity, which were approximately 90% larger in Dahl salt-sensitive and C17S.L2 versus Lewis rats and only 35% to 45% larger in C10S.L16 versus Lewis rats. In another set of rats on regular salt, blood pressure and heart rate were recorded by telemetry before and during intracerebroventricular infusion of Na(+)-rich cerebrospinal fluid for 14 days. Na(+)-rich cerebrospinal fluid caused significantly larger increases in blood pressure and heart rate, larger responses to air stress and more impairment of baroreflex in Dahl salt-sensitive and C17S.L2 rats versus Lewis rats. In contrast, responses in C10S.L16 rats were similar to those in Lewis rats. These data suggest that, in Dahl salt-sensitive rats, genetic variants in C10QTL2 but not C17QTL contribute to increased neuronal responsiveness to cerebrospinal fluid [Na(+)]. However, neither of them contributes to the increase in cerebrospinal fluid [Na(+)] induced by high salt.

Cryopreservation and orthotopic transplantation of rat ovaries as a means of gamete banking

Besides the exponentially increasing number of mouse strains, the rising number of rat strains, due to the establishment of transgenic and coisogenic strains in this species, surpasses the capacity of most animal houses. Cryopreservation of gametes may be a means of solving these problems. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. Ovaries of the rat inbred strain WKY/Ztm were frozen with dimethylsulphoxide as cryoprotectant and stored at -196 degrees C. Orthotopical transplantation was performed into ovariectomized syngenic recipients. Re-establishment of the reproductive cycle in the recipients was confirmed by vaginal cytology. The morphological integrity of frozen and unfrozen ovaries was compared by histological means after staining with haematoxylin and eosin. The number of litters and offspring was recorded. Reproductive cycle was re-established in all recipients of unfrozen ovaries and in more than 50% of recipients that received frozen-thawed ovaries. One-third of the former and more than 25% of the latter became pregnant and delivered at least one litter. Cyropreservation of ovaries can thus be considered as a reliable method of preserving scientifically and economically important mutant stock, as well as congenic rat strains that are currently not required.

Congenic strains confirm aerobic running capacity quantitative trait loci on rat chromosome 16 and identify possible intermediate phenotypes

We previously identified two inbred rat strains divergent for treadmill aerobic running capacity (ARC), the low-performing Copenhagen (COP) and the high-performing DA rats, and used an F(2)(COPxDA) population to identify ARC quantitative trait loci (QTLs) on rat chromosome 16 (RNO16) and the proximal portion of rat chromosome 3 (RNO3). Two congenic rat strains were bred to further investigate these ARC QTLs by introgressing RNO16 and the proximal portion of RNO3 from DA rats into the genetic background of COP rats and were named COP.DA(chr 16) and COP.DA(chr 3), respectively. COP.DA(chr 16) rats had significantly greater ARC compared with COP rats (696.7 +/- 38.2 m vs. 571.9 +/- 27.5 m, P = 0.03). COP.DA(chr 3) rats had increased, although not significant, ARC compared with COP rats (643.6 +/- 40.9 m vs. 571.9 +/- 27.5 m). COP.DA(chr 16) rats had significantly greater subcutaneous abdominal fat, as well as decreased fasting triglyceride levels, compared with COP rats (P < 0.05), indicating that genes responsible for strain differences in fat metabolism are also located on RNO16. While this colocalization of QTLs may be coincidental, it is also possible that these differences in energy balance may be associated with the superior running performance of COP.DA(chr 16) consomic rats.

Triplet repeat in the Repin1 3′‐untranslated region on rat chromosome 4 correlates with facets of the metabolic syndrome

Congenic and subcongenic rat strains confirmed the quantitative trait loci (QTLs) for facets of the metabolic syndrome between 60.53 and 77.11 Mb on chromosome 4. The analysis of candidate genes in this region favoured the replication initiator 1 (Repin1) characterized by a SNP in the coding region and a triplet repeat (TTT) in the 3'-untranslated region (3'UTR). We analysed nine rat strains (BB/OK, SHR, F344, BN, DA, LEW, hHTg, WOKW, and their founders WOK-F) and four wild rats on DNA (sequencing) and RNA level (gene expression in blood, liver, subcutaneous, and epididymal adipocytes). In addition, the rats were phenotypically characterized in order to link the rat phenotype to genotype differences in the QTL on chromosome 4. Wild rats were heterozygous for the SNP (C/T), whereas all the inbred strains were homozygous. The shortest triplet repeat was found in SHR (5) and the highest was found in hHTg and WOKW (11), which developed metabolic disorders. The repeat number correlated with most phenotypic traits studied. Using linear multiple regression analysis with repeat size as the dependent variable and considering all the data of this study, it was clearly demonstrated that not only VLDL cholesterol and serum insulin but also the expression of Repin1 in the liver is significantly associated with the repeat size of the 3'UTR. It is concluded that the triplet repeat expansion in 3'UTR is involved in metabolic alterations as found in hHTg and WOKW rats and that the functional unknown gene, Repin1, could be a novel candidate gene for the development of facets of the metabolic syndrome.

Separately Inherited Defects in Insulin Exocytosis and β-Cell Glucose Metabolism Contribute to Type 2 Diabetes

The effects of genetic variation on molecular functions predisposing to type 2 diabetes are still largely unknown. Here, in a specifically designed diabetes model, we couple separate gene loci to mechanisms of beta-cell pathology. Niddm1i is a major glucose-controlling 16-Mb region in the diabetic GK rat that causes defective insulin secretion and corresponds to loci in humans and mice associated with type 2 diabetes. Generation of a series of congenic rat strains harboring different parts of GK-derived Niddm1i enabled fine mapping of this locus. Congenic strains carrying the GK genotype distally in Niddm1i displayed reduced insulin secretion in response to both glucose and high potassium, as well as decreased single-cell exocytosis. By contrast, a strain carrying the GK genotype proximally in Niddm1i exhibited both intact insulin release in response to high potassium and intact single-cell exocytosis, but insulin secretion was suppressed when stimulated by glucose. Islets from this strain also failed to respond to glucose by increasing the cellular ATP-to-ADP ratio. Changes in beta-cell mass did not contribute to the secretory defects. We conclude that the failure of insulin secretion in type 2 diabetes includes distinct functional defects in glucose metabolism and insulin exocytosis of the beta-cell and that their genetic fundaments are encoded by different loci within Niddm1i.

Effect of angiotensin II blockade on a new congenic model of hypertension derived from transgenic Ren-2 rats

The generation of the Lew.Tg(mRen2) congenic hypertensive rat strain, developed through a backcross of the hypertensive (mRen2)27 transgenic rat with normotensive Lewis rats, provides a new model by which primary hypertension can be studied without the genetic variability found in the original strain. The purpose of this study was to characterize the Lew.Tg(mRen2) rats by dually investigating the effects of type 1 angiotensin II (ANG II) receptor (AT(1)) blockade and angiotensin-converting enzyme (ACE) activity inhibition on the ANG-(1-7)/ACE2 axis of the renin-angiotensin system in this new hypertensive model. The control of blood pressure elicited by 12-day administration of either lisinopril (mean difference change = 92 +/- 2, P < 0.05) or losartan (mean difference change = 69 +/- 2, P < 0.05) was associated with 54% and 33% increases in cardiac ACE2 mRNA and 54% and 43% increases in cardiac ACE mRNA, respectively. Lisinopril induced a 3.1-fold (P < 0.05) increase in renal cortical expression of ACE2, whereas losartan increased ACE2 mRNA 3.5-fold (P < 0.05). Both treatment regimens increased renal ACE mRNA 2.6-fold (P < 0.05). The two therapies augmented ACE2 protein activity, as well as increased cardiac and renal AT(1) receptor mRNAs. ACE inhibition reduced plasma ANG II levels (81%, P < 0.05) and increased plasma ANG-(1-7) (265%, P < 0.05), whereas losartan had no effect on the peptides. In contrast with what had been shown in normotensive rats, ACE inhibition decreased renal ANG II excretion and transiently decreased ANG-(1-7) excretion, whereas losartan treatment was associated with a consistent decrease in ANG-(1-7) urinary excretion rates. In response to the treatments, the expression of both renal cortical renin and angiotensinogen mRNAs was significantly augmented. The paradoxical effects of blockade of ANG II synthesis and activity on urinary excretion rates of the peptides and plasma angiotensins levels suggest that, in Lew.Tg(mRen2) congenic rats, a failure of compensatory ACE2 and ANG-(1-7)-dependent vasodepressor mechanisms may contribute both to the development and progression of hypertension driven by increased formation of endogenous ANG II.

Expression of Hypertension During Development of an Okamoto SHR/Brown Norway Congenic Rat Strain

Expression of Hypertension During Development of an Okamoto SHR/Brown Norway Congenic Rat Strain

Development of Congenic Rat Strains for Alcohol Consumption Derived from the Alcohol-Preferring and Nonpreferring Rats

A genome scan of the F2 generation from an inbred alcohol-preferring (iP) and inbred alcohol-nonpreferring (iNP) rat cross identified a significant quantitative trait locus (QTL) on chromosome 4 with a lod score of 9.2. To confirm this QTL and to create animals for fine mapping of the QTL region, chromosome 4 reciprocal congenic strains were developed by transferring the chromosome 4 QTL interval into the respective iP or iNP backgrounds. The iP strain was crossed with the iNP strain to create iPiNP F1 animals, which were backcrossed to either iNP or iP animals to produce the N2 generation. Using marker-assisted selection, 10 generations of backcrossing were performed. The selection was followed by an intercross between the N10 animals to produce homozygous animals (N10F1), resulting in the finished congenic strains. Congenic strains in which the iP chromosome 4 QTL interval was transferred to the iNP (NP.P) and the iNP chromosome 4 QTL was transferred to the iP (P.NP) exhibited the expected effect on alcohol consumption of the donor strain. Development of these congenic strains further indicates that the chromosome 4 QTL region is, in part, responsible for the disparate alcohol consumption observed between the iP and iNP rats. These congenic animals will be an invaluable resource for fine mapping the QTL region and for the identification of the gene(s) that influences the drinking behavior of the iP and iNP rats.

Gene expression studies support obesity related sub-phenotypes in BB.4S and BB.4W rats

Aim: There are two congenic BB rat strains carring a SHR segment (D4Got41-Tacr1; BB.4S) or a WOKW segment (D4Got41-Fabp1; BB.4W) of chromosome 4 into BB/OK background. Both congenics develop obesity with hyperinsulinemia (BB.4W) or with hyperleptinemia (BB.4S) representing two sub-phenotypes related to obesity. Therefore, we studied the gene expression in adipose tissues and hypothalamus of BB.4S and BB.4W rats.

Localization of Eutr2, a locus controlling susceptibility to DES-induced uterine inflammation and pyometritis, to RNO5 using a congenic rat strain

In some rat strains chronic administration of exogenous estrogens induces pyometritis, an inflammation of the uterus associated with infection, suggesting that there is genetic variation in susceptibility to estrogen-induced inflammation and pyometritis. In this article we report that following 10 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), Fisher 344 (F344) rats exhibit modest uterine inflammation and a 0% incidence of pyometritis. By contrast, under identical experimental conditions, Brown Norway (BN) rats exhibit significant inflammation and a 100% incidence of pyometritis. Similarly, we also observed profound uterine inflammation and a 100% incidence of pyometritis in a congenic rat strain in which a segment of RNO5 from the BN strain is carried on the F344 strain. These data suggest that a locus on RNO5 controls both the magnitude of DES-induced uterine inflammation and susceptibility to DES-induced pyometritis. This locus, designated Eutr2, resides within the same segment of RNO5 as the Eutr1 locus, which confers susceptibility to E2-induced pyometritis in an F2 population generated in a cross between the BN and August x Copenhagen 9935, Irish (ACI) strains.

Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5

In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17beta-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August x Copenhagen Irish (ACI) strain. In a (BN x ACI)F(2) population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN x ACI)F(2) population.

Are IA-2 and RESP18 Involved in Trait of Blood Pressure?

To the Editor: Congenic rat strains are important tools for the genetic dissection of essential hypertension.1 Garrett et al recently demonstrated that a blood pressure (BP) quantitative trait locus (QTL) exists within a newly defined 117-kb QTL region on rat chromosome 9.2 By using microarray technology, the authors first found that the mRNA of Resp18 (endocrine-specific protein 18) is &7.27-fold lower in the kidney of S.R(9)x3A congenic rats (Dahl salt-resistant) than that of S rats (Dahl salt-sensitive). Furthermore, sequencing analysis revealed multiple mutations of Resp18 , particularly the sequence variation (T/C) in exon 2. However, a fine-map analysis showed that Resp18 is located just outside of the 117-kb QTL region; thus, Resp18 was eliminated as a candidate gene. The authors, however, did not address: (1) whether a homolog gene of Resp18 exists in genome, particularly in the 117-kb QTL region; (2) the significance of the Resp18 sequence variation (T/C) in exon 2; and (3) the possibilities that RESP18 plays a role in BP. We recently demonstrated that RESP18 not only shares significant sequence similarities with the N -terminal domain (amino acid 1&200) of IA-2, a dense-core vesicle (DCV)-transmembrane protein, but also shows a similar biological function to IA-2 in …

A speed congenic rat strain bearing the tongue cancer susceptibility locus Tscc1 from Dark-Agouti rats

We previously reported that Dark-Agouti (DA) rats are highly susceptible to 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancer (TC), whereas Wistar/Furth (WF) rats are barely susceptible. Linkage analysis of reciprocal (DA×WF)F2 rats demonstrated five quantitative trait loci, Tongue squamous cell carcinoma 1–5 ( Tscc1–5) determining the size and number of the TCs. The major susceptibility locus Tscc1 is mapped on rat chromosome 19. In the present study, we used a marker-assisted speed congenic procedure to construct WF.DA- Tscc1 (WF-T1D) rats, i.e. WF rats carrying a DA-derived Tscc1 chromosomal segment, and evaluated the effect of a single Tscc1 on 4NQO-induced tongue carcinogenesis. In WF-T1D rats, the incidence, number and size of 4NQO-induced TCs were significantly higher than those in WF rats, indicating that the introgressed segment contains one of the susceptibility loci for 4NQO-induced TCs from DA rats. Detection of a single nucleotide polymorphism in NQO1, one of the Tscc1 candidate genes, enabled us to map NQO1 in the Tscc1 segment between D19Wox8 and D19Wox7 on chromosome 19. Possible relevance of NQO1 polymorphism to TC susceptibility is discussed.

Terminal Complement Components Mediate Release of von Willebrand Factor and Adhesion of Platelets in Arteries of Allografts

Both humoral and cellular immune responses can cause arterial injury in organ transplants, but the manifestations of these different inflammatory mechanisms have not been dissected fully. The present study was designed to define the effects of the terminal complement components on arterial injury in vivo. The authors have developed congenic rat strains with a C6 deficiency. The absence of C6 terminates the cascade of complement after C5 cleavage and prevents the assembly of the membrane attack complex. Hearts were transplanted from PVG.1A (RT1) rats to major histocompatibility complex-incompatible C6-deficient (C6-) or C6-sufficient (C6+) PVG.1U (RT1) rats. PVG.1A (C6-) cardiac grafts were rejected acutely (6-7 days) by untreated PVG.1U (C6+) recipients but survived significantly longer in PVG.1U (C6-) recipients (8 to >30 days). Arteries of cardiac allografts in C6+ recipients demonstrated extensive endothelial injury evidenced by release of von Willebrand factor (vWF) and accompanied by platelet aggregation. In contrast, vWF was retained in Weibel-Palade storage granules of arterial endothelial cells in cardiac allografts that were rejected by C6- recipients. In the absence of C6, intimal alterations were limited to lifting of endothelial cells from supporting stroma by infiltrating mononuclear cells, duplicating the clinical lesion described as endotheliitis or intimal arteritis. Delaying graft rejection with a short course of cyclosporine did not decrease vWF release and platelet aggregation in PVG.1U (C6+) recipients. Mononuclear cell infiltration of the arterial intima occurs in the absence of C6, but C6 deficiency limits the release of vWF from arterial endothelial cells.

Abstracts

Abstracts

A Novel Model of Obesity-Related Diabetes: Introgression of the Leprfa Allele of the Zucker Fatty Rat into Nonobese Spontaneously Diabetic Torii (SDT) Rats

An fa allele of the leptin receptor gene (Lepr(fa)) of the Zucker fatty rat was introduced into the genome of the Spontaneously Diabetic Torii (SDT) rat, an inbred model of nonobese type 2 diabetes mellitus, through the 'Speed congenic method'. The newly established congenic strain of a SDT rat for Lepr(fa) was maintained by intercrossing between fa-heterozygous littermates, and the phenotypes related to obesity and diabetes were investigated till 32 wks of age. SDT fa/fa rats of both sexes exhibited obesity, adiposity and insulin resistance associated with hyperphagia from the loss of leptin action. Interestingly, they developed diabetes from 5 wks of age in males and 8 wks in females with the incidences reaching 100% at 16 wks in males and 73% at 32 wks in females. In contrast, heterozygous (+/fa) and wild-type (+/+) rats developed spontaneous nonobese diabetes in males from approximately 20 wks, but not in females, as with the original SDT rats. These results indicate that the fa gene accelerates the onset of diabetes in SDT rats by making adiposity and/or insulin resistance as potent risk factors for development of their diabetes. The SDT.Lepr(fa) congenic rat strain is expected to be a novel model of obesity-related diabetes and could be a useful tool for studies of the genetic backgrounds of diabetes in response to fa-induced obesity.