No relationship between sequence variation in protein coding regions of the Tas1r3 gene and saccharin preference in rats

Nearly all mammalian species like sweet-tasting foods and drinks, but there are differences in the degree of 'sweet tooth' both between species and among individuals of the same species. Some individual differences can be explained by genetic variability. Polymorphisms in a sweet taste receptor (Tas1r3) account for a large fraction of the differences in consumption of sweet solutions among inbred mouse strains. We wondered whether mice and rats share the same Tas1r3 alleles, and whether this gene might explain the large difference in saccharin preference among rats. We conducted three experiments to test this. We examined DNA sequence differences in the Tas1r3 gene among rats that differed in their consumption of saccharin in two-bottle choice tests. The animals tested were from an outbred strain (Sprague-Dawley; experiment 1), selectively bred to be high- or low-saccharin consumers (HiS and LoS; experiment 2), or from inbred strains with established differences in saccharin preference (FH/Wjd and ACI; experiment 3). Although there was considerable variation in saccharin preference among the rats there was no variation in the protein-coding regions of the Tas1r3 gene. DNA variants in intronic regions were detected in 1 (of 12) outbred rat with lower-than-average saccharin preference and in the ACI inbred strain, which also has a lower saccharin preference than the FH/Wjd inbred partner strain. Possible effects of these intronic nucleotide variants on Tas1r3 gene expression or the presence of T1R3 protein in taste papillae were evaluated in the ACI and FH/Wjd strains. Based upon the results of these studies, we conclude that polymorphisms in the protein-coding regions of the sweet receptor gene Tas1r3 are uncommon and do not account for individual differences in saccharin preference for these strains of rats. DNA variants in intron 4 and 5 are more common but appear to be innocuous.     

Slc:Wistar outbred rats show close genetic similarity with F344 inbred rats

Although Slc:Wistar rats are used widely in biomedical research as outbred rats, close similarities in growth curves, survival rates, and immunological and biochemical phenotypes have been reported between Slc:Wistar and F344 inbred rats. We reported previously that nine genetic variations that were fixed in Slc:Wistar rats had identical genotypes in F344 rats. Here, we examined the genetic characteristics of Slc:Wistar rats using 27 simple-sequence length polymorphism (SSLP) markers and compared them with other Wistar stocks available in Japan and with some F344 strains. Among 27 SSLP loci, 23 (85%) were fixed in the Slc:Wistar rats, which was the highest among the other Wistar stocks. The 23 fixed loci shared identical genotypes with corresponding loci in F344 rats. Further, the predominant allele types in the unfixed loci had allele frequencies as high as 80%, and these alleles were identical in the F344 rats. When the nine genetic variations reported previously are added, a total of 32 (89%) out of the 36 loci examined were fixed and identical in the Slc:Wistar and F344 rat genomes. These findings indicate the low genetic variation in Slc:Wistar rats and the high genetic similarity between the Slc:Wistar and F344 inbred rats. This study demonstrates the importance of characterizing outbred rats and the need to pay ample attention to the genetic characteristics the Slc:Wistar rats for their proper use.     

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.     

A set of highly informative rat simple sequence length polymorphism (SSLP) markers and genetically defined rat strains

Background

The National Bio Resource Project for the Rat in Japan (NBRP-Rat) is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI) strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP) markers and 122 rat strains, which were genotyped by the marker set.

Results

The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers), showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background.

Conclusion

These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL) analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site [1].

     

A new method for identifying informative genetic markers in selectively bred rats

Microsatellite length polymorphisms are useful for the mapping of heritable traits in rats. Over 4000 such microsatellites have been characterized for 48 inbred rat strains and used successfully to map phenotypes that differ between strains. At present, however, it is difficult to use this microsatellite database for mapping phenotypes in selectively bred rats of unknown genotype derived from outbred populations because it is not immediately obvious which markers might differ between strains and be informative. We predicted that markers represented by many alleles among the known inbred rat strains would also be most likely to differ between selectively bred strains derived from outbred populations. Here we describe the development and successful application of a new genotyping tool (HUMMER) that assigns “heterozygosity” (Het) and “uncertainty” (Unc) scores to each microsatellite marker that corresponds to its degree of heterozygosity among the 48 genotyped inbred strains. We tested the efficiency of HUMMER on two rat strains that were selectively bred from an outbred Sprague-Dawley stock for either high or low activity in the forced swim test (SwHi rats and SwLo rats, respectively). We found that the markers with high Het and Unc scores allowed the efficient selection of markers that differed between SwHi and SwLo rats, while markers with low Het and Unc scores typically identified markers that did not differ between strains. Thus, picking markers based on Het and Unc scores is a valuable method for identifying informative microsatellite markers in selectively bred rodent strains derived from outbred populations.     

PCR扩增近交系大鼠微卫星位点DNA多态性的研究

本实验选取大鼠7条染色体上的微卫星位点合成了10对引物,利用聚合酶链反应（PCR）扩增技术对国内北京和哈尔滨等4家单位提供伯6个品系（SHR、SHRSP、LEW、RCS、WKY和F344）的8个近交系大鼠群体进行了DNA多态性分析的研究。结果表明：9个微卫星位点具有显多态性;不同品系个体之间具有多态性;同一群体不同个体之间除SHR（哈）的SMST位点和WKY（哈）的AGT位点出现一定的差异,其他均没有差异;不同地区同一品系的不同个体之间也存在一定的差异。该方法能有效地对近交系与杂交系、品系与品系、品系与亚系加以区分。因此,本实验为开展近交系大鼠遗传作图、基因定位和为实验动物的遗传背景监测提供可靠的信息,为大鼠遗传基因的研究提供了一个快速简例、特异准确的方法。     

Inbred mouse strains and genetic stability: a review

Inbred mice were essential animal models for scientific research during the 20th century and will contribute decisive results in the current and next centuries. Far from becoming an obsolete research tool, the generation of new inbred strains is continuing and such strains are being used in many research fields. However, their genetic properties have been overlooked for decades, although recent research has revealed new insights into their genetic fragility and relative instability. Contrary to what we usually assume, inbred mice are far from being completely isogenic and both single-gene major mutations and polygenic mutational variability are continuously uploading into inbred populations as new sources of genetic polymorphisms. Note that several inbred strains from new major mutations are released every year, whereas small mutations can accumulate up to accounting for a significant percentage of the phenotypic variance (e.g. 4.5% in a recent study on C57BL/6J mice). Moreover, this genetic heterogeneity can be maintained for several generations by heterozygote selection and, if fixed instead of dropping off, genetic drift must be anticipated. The contribution of accidental genetic contamination in inbred strains must also be considered, although its incidence in current breeding stocks should be minimal, or even negligible. This review revisits several relevant topics for current inbred strains, discussing the latest cutting-edge results within the context of the genetic homogeneity and stability of laboratory mice. Inbred mice can no longer be considered as completely isogenic, but provide a remarkably homogeneous animal model with an inevitable moderate-to-low degree of genetic variability. Despite a certain degree of genetic heterogeneity becoming inescapable, inbred mice still provide very useful animal models with evident advantages when compared with outbred, that is, highly variable, populations.     

Enzyme polymorphism in feral, outbred and inbred rats (Rattus norvegicus).

Polymorphism at 25 loci coding for liver enzymes was studied in two feral, three outbred and three inbred rat strains by starch gel electrophoresis. No variation was found at 14 loci, and a low degree of polymorphism was detected at three. Eight loci were polymorphic in more than one population. The average degree of heterozygosity per locus per individual feral rat was 0-07. The degrees of heterozygosity observed in the outbred and inbred strains were lower, from 0-006 to 0-012. Contrary to expectation, the inbred strains were neither monomorphic nor appreciably less heterozygous than the outbred ones. The heterozygosity of the inbred strains was primarily due to two polymorphic loci, G-3-pdh and alpha-Gpdh. The reason for this polymorphism is probably the superior homeostasis of the heterozygotes.     

Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?

There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection.     

Identification, Mapping and Linkage Analysis of Randomly Amplified DNA Polymorphisms in Tetrahymena Thermophila

Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan. These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain). Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3. They map to all five chromosomes and appear to be evenly distributed throughout the genome. Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R, 2L, 3 and 5. The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms.     

Polymorphisms of four hepatic cytochromes P-450 in twenty-eight inbred strains of rat

Two-dimensional gel electrophoresis of hepatic microsomes from phenobarbital-treated animals was used to analyze electrophoretic/regulatory polymorphisms for cytochromes P-450b, P-450e, P-450g, and P-450h in 28 inbred strains of rat. Previous studies with outbred rats revealed the existence of four electrophoretic variants for P-450b, two for P-450e, and three for P-450h as well as two regulatory alleles for P-450g. With the exception of one allozymic form of P-450h, all of these alleles as well as a novel (null) allele for P-450e were found to be homozygous in at least two of the inbred strains tested. Eight phenotypes for combinations of these four cytochromes P-450 were observed. Inbred strains were identified that can be used in studies on the structure/function of unique cytochrome P-450-allozymes and in genetic crosses to map the four distinct cytochrome P-450 genes.This work was supported by National Institutes of Health Biomedical Research Support Grant SO7RR07208.     

Strain differences in testes degeneration, myopathy, and the lymphocyte mitogen response in vitamin E-deficient rats

The differences in sensitivity to vitamin E deficiency were examined in two genetically related inbred strains of rat, the spontaneously hypertensive rat and its genetic ancestor, the Wistar-Kyoto rat, as well as in the outbred Sprague-Dawley strain. The three strains showed differences in growth rate, myopathy, testes degeneration, and immunological responses in response to vitamin E deficiency with the spontaneously hypertensive rat showing the greatest sensitivity to the deficiency.     

Polymorphic microsatellite markers in the outbred CFW and ICR stocks for the generation of speed congenic mice on C57BL/6 background

Reliable definition of the phenotype of particular alleles is carried out in the genetic background of inbred strains. Appearance of mutations in outbred mice therefore requires the generation of congenic mice. The aim of this study was the establishment of a list of polymorphic microsatellite markers which can be used in a polymerase chain reaction (PCR)-based marker-assisted selection protocol (MASP) to allow the use of the two common outbred stocks, CFW and ICR, as donor animals for the fast generation of congenic C57BL/6 mice. The selection of informative microsatellite markers was carried out to provide a simple evaluation of the PCR products by conventional agarose gel electrophoresis. Outbred mice from three suppliers were examined. In total, 153 microsatellite loci were analysed. Here we present 76 and 70 microsatellite markers polymorphic for the outbred ICR and CFW stocks compared to C57BL/6. At least three microsatellite loci per chromosome were chosen as informative markers for the autosomal genome, giving rise to a maximum marker distance of 58 cM. Thus, additional individual markers have to be selected for the respective outbred mouse which is chosen as a donor animal.     

应用微卫星DNA标记对Wistar和SD大鼠封闭群的遗传学研究

封闭群大鼠的遗传质量对其医学生物学实验结果有重要影响,但目前缺乏遗传检测方法和标准.本研究应用6个微卫星标记及其荧光标记一半自动基因分型技术,对北京和上海2家单位分别提供的Wistar和Spague-Darley(SD)大鼠封闭群进行了遗传检测和评估.6个微卫星位点均具有高度多态性,在两大鼠群体共发现等位基因36个,每位点等位基因数5-8个,其多态信息含量(PIC)从0.5892(D11Mgh3)到0.8019(D6Mit1),平均为0.688l.6个位点在Wistar和SD大鼠分别发现25和26个等位基因,其平均期望杂合度分别为O.6260和0.6249.两群体的各组遗传多样性指数间无显著差异.群体间的不同微卫星位点Fst范围0.046l到0.4363.平均为0.2069,表明其遗传分化程度较大;Nei(1972)遗传距离和Nei(1978)无偏遗传距离分别为1.2862和1.2726,表明了2群体之间较大的遗传差异:Hardy-Weinberg平衡检验表明Wistar大鼠在所有检测的6个位点均非常显著偏离Haraly-WeinJaerg平衡,SD大鼠在2个位点(D6Mit1和D11Mgh3)处于遗传平衡状态,且偏离位点均表现为杂合子缺陷.因此研究表明,Wistar和SD大鼠封闭群均具有较好的遗传多样性,且两群体之间有较大的遗传差异和分化程度,分别具有各自不同的遗传特征,偏离Hardy-Weinberg遗传平衡是其繁育过程中较多存在的问题.本研究结果将为两品系大鼠遗传检测方法和标准的建立提供基础资料和依据.     

The genetic variation of compatibility in Biomphalaria glabrata and Schistosoma mansoni

Interactions between different Biomphalaria glabrata stocks and Schistosoma mansoni strains were studied. A series of inbred stocks of B. glabrata were characterized as to genetic variations in susceptibility at different ages to a series of different S. mansoni strains. A series of inbred strains of S. mansoni were characterized as to genetic variations in infectivity for B. glabrata stocks at different ages. Also described is a process of selection for substrains from a single S. mansoni isolate that differ genetically in snail infectivity.     

The choice of animal model and reduction

Careful choice of the animal model is essential, if research is to be conducted efficiently, by using the minimum number of animals in order to provide the maximum amount of information. Inbred strains of rodents provide an excellent way of controlling and investigating genetic variation in characters of interest and in response to experimental treatments. Outbred stocks, in which genetic and non-genetic factors are inextricably mixed, are much less suitable, because random and uncontrolled genetic variation tends to obscure any treatment responses. In some cases, the use of inbred strains has led to major advances in scientific understanding. The specific example given here is in the understanding of host-parasite relationships but, more generally, inbred strains have been of critical importance in research which has resulted in the award of at least 17 Nobel prizes. And yet, despite the extensive literature on the properties and scientific value of inbred strains, many scientists continue to use outbred stocks in the mistaken belief that the use of such animals will, in some mysterious way, make their research more applicable to humans. There is really no evidence that this is so, and there is much evidence that the use of inbred strains has been highly successful in many disciplines.     

Rates of micronuclei induction in different mouse strains

It has previously been shown that the inbred mouse strain MS/Ae was more sensitive in the micronucleus test to several mutagenic agents than outbred mice. To elucidate the possible influence of inbreeding, several inbred strains including MS/Ae, AKR, BALB/c, C57 BR were compared to the two OF1 and NMRI outbred strains. The 3 mutagenic agents MNNG, MMC and MMS all induced a significantly higher number of micronuclei in the MS/Ae strain than in any of the other mouse strains. AKR was especially resistant to the alkylating agents MMS and MNNG. Hence, except for the MS/Ae mouse strain, no inbred strain showed a systematically higher sensitivity than the outbred strains for all of the 3 mutagenic agents used.     

Blood pressure in rats: a comparison of a multifactorial experimental design to measurements in an outbred stock

Systolic blood pressure was measured in males of 8 inbred strains and 1 outbred stock of rats 5 times over a period of 20 min on 5 consecutive days. The strain means ranged between 107.9 mmHg and 149.3 mmHg. The estimated variance between strains (V = 248.7 mmHg) was about 5 times higher than the variance within strains (V = 54.3 mmHg). The intraindividual variance within strains was relatively constant (V = 24.0-37.6 mmHg), while the interindividual variance varied to a great extent (V = 4.5-44.5 mmHg) from strain to strain. The outbred stock showed values of blood pressure and components of variance similar to those of a single inbred strain. Thus, by investigation of a battery of 8 inbred strains in a multifactorial experimental design a greater phenotypic variability due to genetic strain differences is achieved than by measurements in a single outbred stock.     

Malocclusion of the premolar and molar teeth in the guinea pig.

Malocclusions of the premolar and molar teeth of inbred Strain 2/N and Strain 13/N and outbred Dunkin-Harley guinea pigs were examined. A higher incidence of malocclusion observed in the inbred strains suggested a genetic basis for the disorder.     

Positional cloning of the mouse saccharin preference (Sac) locus

Differences in sweetener intake among inbred strains of mice are partially determined by allelic variation of the saccharin preference (Sac) locus. Genetic and physical mapping limited a critical genomic interval containing Sac to a 194 kb DNA fragment. Sequencing and annotation of this region identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial backcrossing of the 194 kb chromosomal fragment containing the Tas1r3 allele from the high-sweetener-preferring C57BL/6ByJ strain onto the genetic background of the low-sweetener-preferring 129P3/J strain rescued its low-sweetener-preference phenotype. Polymorphisms of Tas1r3 that are likely to have functional significance were identified using analysis of genomic sequences and sweetener-preference phenotypes of genealogically distant mouse strains. Tas1r3 has two common haplotypes, consisting of six single nucleotide polymorphisms: one haplotype was found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor responds to sweeteners.