微卫星DNA标记在近交系大鼠HFJ和MIJ遗传监测中的应用

目的 用24对引物对近交系HFJ和MIJ大鼠的微卫星位点进行多态性分析,并选用近交系Lewis和F344大鼠作为对照,进行比较分析.方法 用传统的酚-氯仿法分别提取4个近交系大鼠MIJ、HFJ、Lewis和F344 的基因组DNA,选取大鼠24个微卫星位点,通过PCR扩增,扩增产物经过非变性聚丙烯酰胺凝胶电泳和银染,根据电泳结果,比较分析4种品系近交系大鼠之间微卫星多态性.结果 4种品系及品系内不同个体的近交系大鼠在24个微卫星位点上的扩增产物均出现一个条带,MIJ和HFJ大鼠在品系间和品系内均表现为单态性,同Lewis 和F344的扩增结果比较,14个位点显示多态性,有10个位点显示单态性.结论 两个近交系大鼠品系MIJ和HFJ符合近交系要求,筛选出的14个多态性微卫星位点可用于有关近交系大鼠的遗传背景监测.     

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.     

微卫星DNA监控大鼠近交系的培育

采用微卫星DNA技术来监控大鼠仔代基因状况,选择性地进行交配繁殖,使基因快速纯合,缩短培育新的近交系动物周期。利用PCR扩增30个微卫星DNA位点对封闭群SD和Wistar大鼠交配繁殖的仔代鼠进行微卫星DNA多态性分析,仔代中与母代SD大鼠相似系数高的与中的进行定向交配繁殖。F2代大鼠均为杂合多态的位点,没有纯合位点;到F9代时基因纯合位点达27个,纯合基因位点率为90%。每代相似系数具有不断上升的趋势,上升率为6-20%。采用皮肤移植方法验证了F9代大鼠间无排斥。建立了一种新的快速培育近交系动物的方法。     

Genetic loci for spleen weight and blood pressure in ISIAH rats with inherited stress-induced arterial hypertension

Recently, the important role of the spleen’s function in hypertension development was demonstrated. In this study, the genetic control of absolute and relative spleen weight was investigated to reveal the genetic loci common for spleen traits and for arterial blood pressure at rest and under the emotional stress conditions in ISIAH rats with inherited stress-induced arterial hypertension. The search for genetic loci for absolute and relative spleen weight was performed on 6-month-old F₂ (ISIAH × WAG) hybrid males derived from a cross of hypertensive ISIAH and normotensive WAG rats. One significant QTL mapped on chromosome 1 and 5 suggestive loci were found for relative spleen weight. Four suggestive loci were detected for absolute spleen weight. All detected loci were novel. The significant QTL on chromosome 1 was common for relative spleen weight and arterial blood pressure at rest and under the emotional stress conditions in ISIAH rats. The results suggest that the manifestation of the stress-sensitive arterial hypertension in ISIAH rats may be related to the changes in genetic control of the spleen function.     

金黄地鼠与白化地鼠遗传生化基因位点概貌

目的建立金黄地鼠和白化地鼠遗传生化基因位点。方法选用小鼠和大鼠的遗传生化基因位点,采用蛋白质和同工酶醋酸纤维电泳的方法,对金黄地鼠和白化地鼠进行生化基因位点检测。结果建立了金黄地鼠和白化地鼠25个生化基因位点,分析金黄地鼠和白化地鼠遗传生化基因位点的多态性,为进一步研究金黄地鼠白化突变系的遗传机理奠定基础。结论金黄地鼠生化基因位点存在多态性,白化地鼠与金黄地鼠生化基因位点存在差异。     

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.     

Evidence for a female-specific effect of a chromosome 4 locus on anxiety-related behaviors and ethanol drinking in rats

Previous studies using the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) led to the mapping of two quantitative trait loci, named Ofil1 (on chromosome 4 of the rat) and Ofil2 (on chromosome 7), for open-field inner locomotion, a behavioral index of anxiety. Studies using other strains showed that the region next to Ofil1 influences measures of not only anxiety but also ethanol consumption. In view of the high prevalence of psychiatric disorders such as anxiety and alcoholism, as well as the comorbidity between them, the present study was designed to better characterize the contribution of these two loci to complex emotional and consummatory responses. Rats deriving from an F2 intercross between the LEW and the SHR strains were selected according to their genotype at markers flanking the loci Ofil1 and Ofil2 and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating four genotypic combinations. These selected animals as well as purebred LEW and SHR rats of both sexes were submitted to a battery of tests including measures of locomotor activity, anxiety, sweet and bitter taste reinforcement and ethanol intake. Lewis rats displayed more anxiety-like behavior and less ethanol intake than SHR rats. Ofil1 (on chromosome 4) affected both the activity in the center of the open field and ethanol drinking in females only. These results suggest that Ofil1 contains either linked genes with independent influences on anxiety-related responses and ethanol drinking or a pleiotropic gene with simultaneous effects on both traits.     

A search for genetic loci responsible for emotional stress-induced arterial hypertension in the ISIAH rats

Hypertension is a widespread human disease caused by a complex interaction of a series of the genetic factors with both each other and the environmental conditions. In this study we aimed at determining the candidate genetic loci responsible for hypertension in the ISIAH rats and studying the dynamics of the relevant genetic and physiological mechanisms in rat ontogeny. The candidate genetic loci were identified from association of the microsatellite markers linked to these loci with arterial hypertension in rat F2 hybrids exposed to stress. Two populations of F2 hybrids of different age (3-4 and 6 months) were obtained by crossing hypertensive ISIAH and normotensive WAG rats. We present the results of cosegregation analysis for the following loci: the gene for the Na+, K(+)-ATPase alpha 1 subunit isoform (Atp1a1), the endothelin-2 gene (Edn2), the low affinity nerve growth factor receptor gene (Lngfr), and a region of chromosome 10 marked with the D10Rat58 microsatellile located 3 cM away of the aldolase C gene (AldC). The results obtained allowed us to localize the genes responsible for the stress-induced arterial hypertension in the ISIAH rats to the Atp1a1 locus (P < 0.05), chromosome 2 and to the Lngfr gene locus (P < 0.05), chromosome 10. The association of hypertensive status with the Lngfr gene was found only in young ISIAH rats whereas in adult rats of this line, hypertension was associated with the Atp1a1 locus.     

Genetic polymorphism of ceruloplasmin in the rat

The genetic heterogeneity of ceruloplasmin in serum was studied in the progeny of the (LEW X BN)F1 X (LEW X BN)F1 rats. The results of the statistical analysis showed that the Hbb and c loci were linked. However, the autosomal Ces gene was not linked to the Hbb or c loci. The Ces gene was expressed in normal Mendelian pattern and had two alleles that manifested a low (Cesl) or high (Cesh) level of the enzyme in serum. The Cesl gene was expressed as a dominant in the F1 generation. Two phenotypes CES-H and CES-L were found in the F2 rats; the females in the parental strain and hybrid had higher ceruloplasmin concentration than the male rats.     

Construction of a high-throughput rat genetic mapping system with 466 arbitrarily primed-representational difference analysis markers

Linkage mapping of quantitative trait loci (QTLs) requires genetic markers that can be efficiently genotyped for a large number of individuals. To isolate genetic markers suitable for this purpose, we previously established the arbitrarily primed RDA (AP-RDA) method. Dot-blotting AP-PCR products (AP-amplicons) onto filters at a high density and hybridization of the filters with the AP-RDA markers made it possible to genotype a large number of individuals simultaneously for multiple loci. In this study, by using 25 primers or primer combinations, we isolated a total of 419 AP-RDA markers by subtracting the AP-amplicon of BUF rats from that of ACI rats, and vice versa. By combining 47 previously isolated markers, a rat genetic map was drawn with 466 AP-RDA markers. Between two given strains of rats other than ACI and BUF, the average informativeness of the markers was 38%. As for the intercross of ACI and BUF rats, 12 selected primers served to genotype 259 loci. In addition, the amounts and quality of genomic DNA to be used for AP-PCR were examined to guarantee reliable genotyping. Now, initial genome scanning of the rat for linkage analysis can be performed efficiently using this mapping system with AP-RDA markers. Received: 28 April 2000 / Accepted: 14 June 2000     

Localization of the di gene in the fourth linkage group in Rattus norvegicus rats

Based on the backcross progeny analysis of rats Rattus norvegicus matings (August X Brattleboro)F1 X Brattleboro, the gene di has been localized in the fourth linkage group at a distance of 26.8 +/- 1.7 cM from the non-agouti loci and 11.4 +/- 4.7 cM from the Svp-1 loci. The gene order proposed is a--Svp-1--di.     

Adoptive immunotherapy of leukemia in the rat, without graft-VS-host complications.

PVG rats bearing a transplantable T cell leukemia were treated with large inocula of lymphoid cells from AUG rats sensitized either against the leukemia or against PVG lymphocytes. AUG and PVG bear identical Ag-B antigens but differ at minor loci, including the Pta loci, which code for differentiation antigens expressed only on peripheral T lymphocytes. Treatment with AUG cells immune to either the PVG leukemia or normal PVG cells resulted in prolonged survival of leukemic rats, a profound but ephemeral leukopenia and prolonged disappearance of leukemic cells from lymphoid tissue. All treated animals, however, eventually died with large, discrete deposits of leukemic cells in both hard and soft tissues. Despite the deliberate mismatching of host and donor cells for minor transplanation antigens, no evidence of GVH symptoms was observed in treated rats. This was interpreted as a result of directing the adoptive immune response to antigens of restricted distribution, i.e., on leukocytes and not on somatic cells.     

Concordance of murine quantitative trait loci for salt-induced hypertension with rat and human loci

To investigate the genetic control of salt-induced hypertension, we performed a quantitative trait locus analysis on male mice from a reciprocal backcross between the salt-sensitive C57BL/6J and the normotensive A/J inbred mouse strains after they were provided with water containing 1% salt for 2 weeks. Genome-wide scans performed on these mice and analyzed with a combination of conventional marker-based regressions and a novel simultaneous search for pairs revealed six significant quantitative trait loci associated with salt-induced blood pressure, two of which were interacting loci. These six loci, named Bpq1-6 for blood pressure quantitative trait loci, mapped to D1Mit334, D1Mit14, D4Mit164, D5Mit31, D6Mit15, and D15Mit13. Furthermore, five of these six loci were concordant with hypertension loci in rats, and four were concordant with hypertension loci in humans, suggesting that quantitative trait loci mapping in model organisms can be used to guide the search for human blood pressure genes.     

Phenotypic selection: a successful strategy to fix major genes of hypertension

Spontaneously diabetic BB/OK rats are not genetically susceptible to develop diabetic complications as hypertension or nephropathy. Recently, we generated 5 congenic BB. SHR rat strains by transferring different chromosomal regions of the spontaneously hypertensive rat (SHR) onto the genetic background of BB/OK rats. Four out of 5 strains showed a weak increase of blood pressure (8 mmHg). This weak blood pressure effect indicated that the transferred regions fo not contain major genes for hypertension. That prompted us to choose the classical procedure of phenotypic selection to fix major genes causing hypertension in a BB/OK rat subline generated by cross of BB/OK and SHR and repeated backcrossing of animals with highest blood pressure onto normotensive BB/OK rats. After 7 backcrosses (N8), all backcross parents were genetically analysed with the aid of 259 microsatellites to identify loci causing blood pressure of 177 ± 10 mmHg in this BB/OK rat subline. The data revealed, that loci on chromosome 1, 14 and 18 were heterozygous until BC5, BC6 and BC7, respectively. Considering the relative stable high blood pressure during the backcross procedure, these loci might be of essential importance for the development of hypertension in the SHR.     

Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition

The different frequency of hepatocellular carcinoma (HCC) in humans at risk suggests a polygenic predisposition. However, detection of genetic variants is difficult in genetically heterogeneous human population. Studies on mouse and rat models identified 7 hepatocarcinogenesis susceptibility (Hcs) and 2 resistance (Hcr) loci in mice, and 7 Hcs and 9 Hcr loci in rats, controlling multiplicity and size of neoplastic liver lesions. Six liver neoplastic nodule remodeling (Lnnr) loci control number and volume of re-differentiating lesions in rat. A Hcs locus, with high phenotypic effects, and various epistatic gene-gene interactions were identified in rats, suggesting a genetic model of predisposition to hepatocarcinogenesis with different subset of low-penetrance genes, at play in different subsets of population, and a major locus. This model is in keeping with human HCC epidemiology. Several putative modifier genes in rodents, deregulated in HCC, are located in chromosomal segments syntenic to sites of chromosomal aberrations in humans, suggesting possible location of predisposing loci. Resistance to HCC is associated with lower genomic instability and downregulation of cell cycle key genes in preneoplastic and neoplastic lesions. p16(INK4A) upregulation occurs in susceptible and resistant rat lesions. p16(INK4A)-induced growth restraint was circumvented by Hsp90/Cdc37 chaperons and E2f4 nuclear export by Crm1 in susceptible, but not in resistant rats and human HCCs with better prognosis. Thus, protective mechanisms seem to be modulated by HCC modifiers, and differences in their efficiency influence the susceptibility to hepatocarcinogenesis and probably the prognosis of human HCC.     

Inheritance of resistance to promotion of preneoplastic liver lesions in Copenhagen rats

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7- (GST 7-7) positive liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. The objective of this study was to determine if resistance is inherited in a dominant or recessive manner and to derive an estimate of the number of genetic loci involved. We crossed male and female Cop rats with F344 rats to produce F1 offspring. Backcross rats were generated using female F1 rats and either Cop or F344 males, resulting in B1c and B1f generations, respectively. The male rats from all these crosses were initiated with diethylnitrosamine (200 mg/kg) at 7 to 8 weeks of age and were promoted 3 weeks later with the RH protocol (2-acetylaminofluorene and a two-thirds partial hepatectomy). The rats were sacrificed 3 weeks after the partial hepatectomy and their livers were sectioned and stained for GST 7-7-positive lesions. The susceptibility of F1 rats was in between Cop and F344 rats, having 21.7% +/- 2.0% (mean +/- SEM) of their liver volume occupied by lesions versus 4.2% +/- 0.8% for Cop and 53.0% +/- 5.8% for F344 rats. As expected, B1c rats had a volume of liver occupied by lesions that was in between the F1 and Cop rats at 13.5% +/- 1.6%. Surprisingly, B1f rats were similar to B1c rats in their resistance (9.1% +/- 2.1%). These results point to a complex, polygenic inheritance pattern that can be explained by a minimum of four loci, one of which shows recessive epistasis.     

Effect of the di mutation (non-sugar diabetes) on properties of kidney proteins in rats

Renal proteins were studied in Brattleboro rats of the didi genotype and in Wistar and Sprague-Dawley rats with normal alleles +2 of this loci. In animals of the +2 genotype maintained under conditions of water deprivation, the content of 120 kDa protein increased significantly in inner medulla of the kidney over 3 days. No changes in the amount of this protein were observed in rats of the didi genotype under the same conditions. We suggest that the congenital inability of didi mutants to synthesize vasopressin accounts for the distinctions observed in the reactions of rats with different genotypes.     

Mapping of two genetic loci,Ten-1 and Ten-2, associated with thymus enlargement in BUF/Mna rats

The thymoma-prone BUF/Mna rat is a useful model for human thymoma. Thymoma develops spontaneously in these rats at an incidence of nearly 100%. At pre-thymoma age, BUF/Mna rats have extremely large thymuses when compared with those of rats of the other strains, suggesting the presence of genes that regulate the thymus enlargement. We performed linkage study to identify the genetic loci associated with thymus enlargement in {(WKY/NCrj × BUF/Mna) F₁× BUF/Mna} backcross rats. Linkage study showed the significant associations between thymus size and markers on Chromosomes (Chrs) 1 and 13, suggesting the presence of two genes, Ten-1 and Ten-2, which regulate the thymus enlargement. Ten-1 was located between myosin light chain, muscle 2 (MYL2) and D1Mgh11 loci on Chr 1, and Ten-2 was located between synaptotagmin II (SYT2) and D13N2 loci on Chr 13. Received: 6 November 1995 / Accepted: 29 March 1996     

Transplantation of metanephroi across the major histocompatibility complex in rats

To determine whether transplanted metanephroi grow, differentiate, and function in hosts that differ in major histocompatibility complex loci (RT1 loci in rats) from donors in a defined way, we implanted metanephroi from embryonic day (E) 15 PVG (RT1(c)) rat embryos into the omentum of nonimmunosupressed uninephrectomized PVG-RT1(avl) (host) rats. By 4 wk posttransplantation, metanephroi had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts had normal structure and ultrastructure. At 12 wk posttransplantation, weights of metanephroi were 54 +/- 8 mg. Inulin clearances were 0.9 +/- 0.3 microl. min(-1). 100 g rat wt(-1). In vitro, splenocytes from PVG rats stimulated the proliferation of cells originating from both PVG-RT1(avl) rats in which a transplant had been performed and PVG-RT1(avl) rats with no transplant. Full-thickness PVG-RT1(avl) skin engrafted normally on PVG-RT1(avl) rats in which PVG metanephroi had been previously implanted and metanephroi retained a normal appearance. In contrast, skin from PVG rats sloughed, and the tubular architecture of metanephroi was obliterated by a mononuclear cell infiltrate consistent with acute rejection. Here we show for the first time that functional chimeric kidneys develop from metanephroi transplanted across the MHC into nonimmunosupressed hosts and provide evidence that a state of peripheral immune tolerance secondary to T cell "ignorance" permits their survival.     

Marker-assisted dissection of genetic influences on motor and neuroendocrine sensitization to cocaine in rats

This study investigated genetic influences on behavioral and neuroendocrine responses to cocaine sensitization. We used male and female rats of the inbred strains Lewis (LEW) and spontaneously hypertensive rats (SHR), which display genetic differences in stress-related responses. The influence of two quantitative trait loci (QTL; Ofil1 and Ofil2 on chromosomes 4 and 7), which modulate stress reactivity in rats, on the effects of cocaine was also investigated through the use of recombinant lines (derived from a LEW   ×   SHR intercross) selected by their genotype at Ofil1 and Ofil2 . Animals were given repeated cocaine or saline injections and tested for locomotion (induction of sensitization). Two weeks later, all animals were challenged with cocaine, and locomotion and corticosterone levels were measured (expression of sensitization). Results indicated that male SHR rats showed more behavioral sensitization than LEW rats, whereas no strain differences in sensitization were seen among females. When challenged with cocaine, LEW and SHR rats of both sexes pretreated with cocaine showed behavioral sensitization compared with saline pretreated animals; however, only LEW rats displayed an increase in the corticosterone levels. Ofil1 was found to influence the induction of sensitization in males and Ofil2 modulated the locomotor effect of cocaine in females. This study provides evidence of a genotype-dependent relationship between the induction and expression of cocaine sensitization, and between the behavioral and neuroendocrine responses induced by cocaine. Moreover, the Ofil1 and Ofil2 loci may contain one or more genes that control the behavioral effects of cocaine in rats.