Assessment of retinal degeneration in outbred albino mice

Evaluation of a pharmaceutical's safety includes assessment of the potential for ophthalmologic toxicity. These nonclinical studies commonly use various outbred stocks of mice. Pretest indirect ophthalmoscopic examinations in the commonly used outbred stock Hsd:ICR(CD-1) indicated that retinal degeneration was a problem in this particular outbred stock of mice. This prompted the authors to examine other stocks of outbred mice routinely used in the performance of nonclinical safety studies. Groups of mice were observed over a 13-week period to determine the progression and changing incidence of retinal degeneration. Light intensity in the room and caging was measured during the study, and it was determined that light did not play a direct role in the progression of the retinal degeneration observed during the study. Histomorphologic examination of the mouse eyes was performed at the end of the study to confirm the presence of retinal degeneration observed after ophthalmoscopic examination. The incidence of retinal atrophy in the various outbred stocks of mice was: Crl:CFW(SW)BR (98.3%), Tac(SW)fBR (80%), Tac:Icr:Ha(ICR)fBR (75%), Hsd:ICR(CD-1) (43.3%), and Crl:CF-1BR (3.0%). Retinal atrophy was not observed in the following outbred mice stocks: Crl:CD-1(ICR)BR, HsdWin:CFW1, and Hsd:NSA(CF-1). On the basis of these findings, it is highly recommended that pretest ophthalmologic screening be performed on mice to obviate pre-existing conditions from confounding or invalidating nonclinical study results.     

Microsatellite-directed selection of breeders for the next backcross generation by using a minimal number of loci

To establish a minimal number of markers for direct selection of candidate mice used for the next mating to produce congenic mice, recombination frequencies of 53 microsatellite loci on chromosomes (chr.) 1 and 19 were examined using 41 N2 mice: the donor strain was BALB/c, and recipient strain was C57BL/6J (B6J) or C57BL/6N (B6N). These markers were spaced at 0.1 to 24.2 centimorgans (cM). Among the 41 mice, B6/B6 homozygosity ranged from 18 to 24 animals (mean, 20; 2 standard deviations, 1.36) for a given locus. There was no difference in recombination frequency between chr. 1 and 19. The recombination frequency of B6J was higher than that of B6N (P < 0.05). Various densities of markers, 10 (5 markers/chr.), 8 (4 markers/chr.), and 6 (3 markers/chr.) spaced at 12.0 to 29.3, 9.0 to 45.0, and 24.5 to 53.0 cM, respectively, were selected from the 53 markers, and homozygosity was compared in each mouse. In mice with decreased homozygosity when tested using 53 markers, homozygosity differed depending on the density of the markers. The results suggested that 3 markers/chr. are sufficient for selection of the highest percentages of homozygosity but are not suitable to define mice with lower percentages of homozygosity.     

A panel of 20 highly variable microsatellite polymorphisms in rhesus macaques (Macaca mulatta) selected for pedigree or population genetic analysis

This paper reports 20 new microsatellite loci that are highly polymorphic in rhesus macaques (Macaca mulatta). We screened known human microsatellite loci to identify markers that are polymorphic in rhesus macaques, and then selected specific loci that show substantial levels of heterozygosity and robust, reliable amplification. The 20 loci reported here were chosen to include one highly informative microsatellite from each rhesus monkey autosomal chromosome. Fourteen of the 20 polymorphisms are tetranucleotide repeats, and all can be analyzed using standard PCR and electrophoresis procedures. These new rhesus markers have an average of 15.5 alleles per locus and average heterozygosity of 0.83. This panel of DNA polymorphisms will be useful for a variety of different genetic analyses, including pedigree testing, paternity analysis, and population genetic studies. Many of these loci are also likely to be informative in other closely related Old World monkey species.     

Genetic analysis of the diabetes-prone C57BLKS/J mouse strain reveals genetic contribution from multiple strains

The C57BLKS/J (BKS) inbred mouse strain is a widely used animal model of type 2 diabetes. In the presence of the diabetes (db) mutation, obese BKS-db mice develop severe diabetes. Genetic studies of diabetes-susceptibility in this strain are facilitated by the fact that BKS is a genetic composite between the diabetes-resistant C57BL/6J (B6) and susceptible DBA/2J (DBA) strains. On this basis, it has been hypothesized that diabetes-susceptibility in BKS is conferred by DBA-derived alleles. However, recent studies revealed non-B6/non-DBA genetic material in BKS. To identify the origin of this genetic component, we generated a genomic map of BKS using 537 microsatellite markers. Our results demonstrate that, in addition to B6 and DBA, BKS contains alleles from at least three other strains, including 129, C57BL/10 and an unidentified mouse strain. We also analyzed two congenic strains, B6-db and BKS-db, which are widely used for the genetic mapping of diabetes-susceptibility loci. We identified several donor-derived genomic regions introduced during the generation of these congenic strains. In summary, our study reveals novel aspects of the genetic fine-structure of BKS and related strains and facilitates the identification of diabetes-susceptibility loci in this mouse model.     

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.     

Major histocompatibility complex heterozygosity reduces fitness in experimentally infected mice

It is often suggested that heterozygosity at major histocompatibility complex (MHC) loci confers enhanced resistance to infectious diseases (heterozygote advantage, HA, hypothesis), and overdominant selection should contribute to the evolution of these highly polymorphic genes. The evidence for the HA hypothesis is mixed and mainly from laboratory studies on inbred congenic mice, leaving the importance of MHC heterozygosity for natural populations unclear. We tested the HA hypothesis by infecting mice, produced by crossbreeding congenic C57BL/10 with wild ones, with different strains of Salmonella, both in laboratory and in large population enclosures. In the laboratory, we found that MHC influenced resistance, despite interacting wild-derived background loci. Surprisingly, resistance was mostly recessive rather than dominant, unlike in most inbred mouse strains, and it was never overdominant. In the enclosures, heterozygotes did not show better resistance, survival, or reproductive success compared to homozygotes. On the contrary, infected heterozygous females produced significantly fewer pups than homozygotes. Our results show that MHC effects are not masked on an outbred genetic background, and that MHC heterozygosity provides no immunological benefits when resistance is recessive, and can actually reduce fitness. These findings challenge the HA hypothesis and emphasize the need for studies on wild, genetically diverse species.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

不同品系小鼠对代谢性高尿酸血症造模的影响

目的：分别以昆明种小鼠及ICR、C57BL/6J小鼠为研究对象,比较在复制高尿酸血症模型时可能的小鼠品系差异,并通过降尿酸药物别嘌呤醇与非布索坦验证选择降尿酸药物筛选时选用不同品系动物造模的影响。方法：采用不同剂量次黄嘌呤腹腔注射联用尿酸酶抑制剂氧嗪酸钾皮下注射给药,测定不同造模时段各品系小鼠血清尿酸值。结果：ICR、C57BL/6J小鼠对高尿酸血症造模耐受显著高于昆明种小鼠,在腹腔注射次黄嘌呤500mg/kg,皮下注射氧嗪酸钾300mg/kg时,才可获得稳定的可用于药物筛选的高尿酸血症模型。结论：选择高尿酸血症在体模型时,昆明种小鼠灵敏度高于ICR小鼠以及近交系的C57BL/6J小鼠。     

Isolation of microsatellite markers from Pinus densiflora Sieb. et Zucc. using a dual PCR technique

We developed seven microsatellite loci from Pinus densiflora using a dual polymerase chain reaction (PCR) technique. Of 186 clones from a library based on suppression PCR, 127 contained microsatellite sequences. Of these, 43 candidates were determined sequences of both flanking regions, and 16 regions from this group were chosen as development markers. Seven of these primer pairs successfully amplified polymorphic single loci among 83 resistant trees against pine wood nematode. The observed heterozygosity of the seven microsatellite markers ranged from 0.247 to 0.843. Mendelian inheritance was confirmed using megagametophytes.     

A genome-wide set of congenic mouse strains derived from CAST/Ei on a C57BL/6 background

We previously reported the construction of two sets of heterozygous congenic strains spanning the mouse genome. For both sets, C57BL/6J was employed as the background strain while DNA from either DBA/2 or CAST/Ei was introgressed to form the congenic region. We have subsequently bred most of these strains to produce homozygous breeding stocks. Here, we report the characterization of the strain set based on CAST/Ei. CAST/Ei is the most genetically distant strain within the Mus mus species and many trait variations relevant to common diseases have been identified in CAST/Ei mice. Despite breeding difficulties for some congenic regions, presumably due to incompatible allelic variations between CAST/Ei and C57BL/6, the resulting congenic strains cover about 80% of the autosomal chromosomes and will be useful as a resource for the further analysis of quantitative trait loci between the strains.     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

High-resolution genetic mapping of the sucrose octaacetate taste aversion (Soa) locus on mouse Chromosome 6

An acetylated sugar, sucrose octaacetate (SOA), tastes bitter to humans and has an aversive taste to at least some mice and other animals. In mice, taste aversion to SOA depends on allelic variation of a single locus, Soa. Three Soa alleles determine `taster' (Soa <sup>a</sup> ), `nontaster' (Soa ^b ), and `demitaster' (Soa ^c ) phenotypes of taste sensitivity to SOA. Although Soa has been mapped to distal Chromosome (Chr) 6, the limits of the Soa region have not been defined. In this study, mice from congenic strains SW.B6-Soa ^b , B6.SW-Soa ^a , and C3.SW-Soa ^a/c and from an outbred CFW strain were genotyped with polymorphic markers on Chr 6. In the congenic strains, the limits of introgressed donor fragments were determined. In the outbred mice, linkage disequilibrium and haplotype analyses were conducted. Positions of the markers were further resolved by using radiation hybrid mapping. The results show that the Soa locus is contained in a ∼1-cM (3.3–4.9 Mb) region including the Prp locus. Received: 5 February, 2001 / Accepted: 1 May, 2001     

不同群系小鼠胚胎玻璃化冷冻保存技术的研究

利用 EFS40 ,二步法对近交系 C5 7BL/6、DBA/2和远交群 ICR小鼠囊胚玻璃化冷冻保存 ,并对冷冻后胚胎体内、外发育效果进行比较。结果表明 ,相同条件下鲜胚经培养 ,近交系 C5 7BL /6小鼠的囊胚发育率 ( 93% )与 ICR( 1 0 0 % )相比差异不显著 ( P>0 .0 5 ) ;而两近交系的囊胚孵化率明显低于 ICR( P<0 .0 1 )。 C5 7BL/6、DBA/2小鼠囊胚冷冻后发育率 ( 93% ,96% )和孵化率 ( 5 2 % ,46% )与各自对照组 ( 1 0 0 % ,1 0 0 %和 61 % ,62 % )相比均无显著差异 ( P>0 .0 5 ) ;并且与 ICR冷冻组发育率和孵化率 ( 94% ,5 3% )之间也无显著差异 ( P>0 .0 5 )。两近交系冻胚移植妊娠与各自对照组和 ICR冷冻组比较均无显著差异 ( P>0 .0 5 )。C5 7BL/6胚胎移植产仔率 ( 35 % )与对照组 ( 5 1 % )之间差异显著 ( P<0 .0 1 ) ,而 DBA/2胚胎移植产仔率 ( 4 7% )与对照组和 ICR冷冻组 ( 39% ,5 8% )相比差异不显著 ( P>0 .0 5 )。     

The<Emphasis Type="Italic"> Sr1</Emphasis> gene that controls diversity of the anti-inulin antibody response maps to mouse chromosome 14

Previous studies demonstrated that the diversity of the antibody response of mice to the inulin (In) determinant of bacterial levan is regulated by the gene Spectrotype Regulation 1 ( Sr1). BALB/c mice produce a monoclonal anti-In response as shown by isoelectric focusing analysis. In contrast, the anti-In antibody response of (BALB/cxC57BL/6)F1 mice is significantly more heterogeneous. We performed a backcross and a genome-wide scan with microsatellite markers and found that Sr1 is tightly linked to D14Mit121 on chromosome (Chr) 14. This location for Sr1 was supported by analysis of CXB Recombinant Inbred strains. We further confirmed this by finding that the Chr 14 congenic mouse strain B6.C-H8 lacks the C57BL/6 allele of the Sr1 gene, indicating that Sr1 is located in the segment of Chr 14 replaced with BALB/c donor DNA. These data place Sr1 near to or coincident with the Tcra/Tcrd T-cell receptor gene complex and suggest a role for T cells in diversifying the anti-In response.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

An investigation of genetic variation within a series of congenic strains of mice

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.     

Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus.

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.     

Mapping of jog locus to the region between D6Mit104 and D6Mit336 on mouse chromosome 6.

The joggle mouse is a recessive ataxic mutant carrying an unknown mutation in a C3H/He (C3H)-derived chromosomal segment. Taking advantage of the mouse genome database, we selected 127 DNA microsatellite markers showing heterozygosity between C3H and C57BL/6J (B6) and a first round of screening for the joggle mutation was performed on B6-jog/+ partial congenic mice (N4). We identified 4 chromosomal regions in which 13 microsatellite markers show heterozygosity between C3H and B6. Then, we analyzed the genotype of these 4 chromosomal regions in mice that showed the joggle phenotype and mapped the jog locus between markers D6Mit104 (111.4 Mb) and D6Mit336 (125.1 Mb) (an interval of 13.7 Mb) on chromosome 6. By using a partial congenic strain together with the mouse genome database, we successfully mapped the chromosomal localization of the jog locus much more efficiently than by conventional linkage analysis.     

Mapping of genes involved in murine herpes simplex virus keratitis: identification of genes and their modifiers

Herpes simplex keratitis (HSK) is an inflammatory response to viral infection and self antigens in the cornea and is a major cause of blindness. Using two strains of mice which are susceptible (129/SVEV) and resistant (C57BL/6) to herpes simplex virus (HSV) strain KOS, (129/SVEV x C57BL/6)F(2) mice were generated and examined for their disease susceptibility in terms of clinical symptoms, ocular disease, and antibody production following corneal scarification with HSV (KOS). A genome-wide screen was carried out using microsatellite markers to determine the genetic loci involved in this response. Loci on chromosomes 4, 5, 12, 13, and 14 were shown to be involved in general susceptibility to clinical disease, whereas loci on chromosomes 10 and 17 were shown to be unique to ocular disease.     

The Inbred Mouse in Pigmentation Research:

Pigment mutations in inbred mice have been important to many new scientific developments over the past century. Inbred mice are essentially genetically alike because of 10–20 generations or more of sibling mating or the equivalent. Mice of the same inbred strain that differ at only one locus can be used to evaluate the phenotypic effects of that one locus without complication of variation at other loci. Similarly, genic interactions among the functions of two or more loci are evaluated by comparing them in all combinations against a uniform genetic background. The next logical step in describing the pigment system will occur when all pigment cell biologists who use mice (cells, tissues, DNA, RNA) make certain that their mice are congenic with C57BL/6J. As a result, the work of all investigators will be genetically comparable. Their work will also be comparable to those investigating other organ systems, because NIH has chosen C57BL/6J as one of its two standard strains. As a result of this standardization, interactions among the different gene loci that function in the pigment system will become more readily evident and the community of pigment cell biologists using congenic mice will be able to analyze the functional interplay of loci that regulate the entire pigment system in the same way that earlier researchers analyzed one mutant allele, or the interactions of two mutant loci.