A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome

Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.     

Identification of a genetic contamination in a commercial mouse strain using two panels of polymorphic markers

Rapid detection of genetic contamination is critical in mouse studies involving inbred strains. During a Quantitative Trait Locus (QTL) study using simple sequence length polymorphism (SSLP) markers, we noticed heterozygosity at some loci of a commercially available inbred C57BL/6N mouse strain, suggesting a contamination by another mouse strain. A panel of 100 single-nucleotide polymorphism (SNP) markers was used to confirm and specify the genetic contamination suspected. Retrospective analyses demonstrated that the contamination took place as early as autumn 2003 and has persisted ever since at a fairly constant level. Contaminating alleles most probably originated from a DBA strain. Our data demonstrate the suitability of SNP markers for rapid detection and identification of the source of genetic contamination. Further, our results show the importance of a state-of-the-art genetic monitoring of the authenticity of murine inbred strains.     

New murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR and mapped by use of recombinant inbred strains

Oligonucleotide primers of random sequence that were 12 bases in length, 58% in GC content, and lacking internal palindromes were designed. By random amplified polymorphic DNA (RAPD) PCR, these primers were used to survey for DNA variations between the progenitors of the mouse AXB and BXA recombinant inbred sets (A/J and C57BL/6J). We identified 17 DNA variants detected by 10 primers. Map positions for these variants were determined by comparing their strain distribution patterns in the AXB, BXA recombinant inbred sets with strain distribution patterns of previously published loci. When necessary, BXD and NXSM recombinant inbred sets were also used. These 17 new loci mapped to 12 chromosomes. The 10 primers were also used to survey 20 inbred mouse strains including the progenitors of other recombinant inbred sets and four mouse strains recently inbred from the wild (CAST/Ei, MOLF/Ei, PERA/Ei, and SPRET/Ei).     

Assignment of the structural gene for argininosuccinate synthetase to proximal mouse chromosome 2

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-1), we have searched for restriction fragment length polymorphisms in the mouse genome using cloned sequences from the mouse arginosuccinate synthetase structural gene. Five restriction fragment length polymorphisms were found among the recombinant inbred progenitor strains AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J. Of these, four polymorphisms were found to distinguish the SWR/J strain from the other six strains, which all had the same fragment. The fifth polymorphism revealed differences among the progenitor strains for recombinant inbred strain sets AKXL, BXD, and SWXL. The strain distribution pattern for this polymorphism indicated close linkage of Ass-1 to Hc (the fifth component of complement) on proximal mouse chromosome 2 with a recombination fraction of 0.016 and a 95% confidence interval of 0.003 to 0.054. These data place Ass-1 in a syntenic group with the genes Hc, Abl, Fpgs, and Ak-1 whose linkage has been conserved between human chromosome 9q and mouse chromosome 2.     

PCR-analyzed microsatellites of the mouse genome—additional polymorphisms among ten inbred mouse strains

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphims by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35–60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.     

Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner

ABSTRACT: BACKGROUND: There is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. Using genetically diverse but fully inbred strains of mice it has been shown that host sequence variants have a strong influence on the severity of influenza A disease progression. In particular, C57BL/6 J, the most widely used mouse strain in biomedical research, is comparatively resistant. In contrast, DBA/2 J is highly susceptible. RESULTS: To map regions of the genome responsible for differences in influenza susceptibility, we infected a family of 53 BXD-type lines derived from a cross between C57BL/6 J and DBA/2 J strains with influenza A virus (PR8, H1N1). We monitored body weight, survival, and mean time to death for 13 days after infection. Qivr5 (quantitative trait for influenza virus resistance on chromosome 5) was the largest and most significant QTL for weight loss. The effect of Qivr5 was detectable on day 2 post infection, but was most pronounced on days 5 and 6. Survival rate mapped to Qivr5, but additionally revealed a second significant locus on chromosome 19 (Qivr19). Analysis of mean time to death affirmed both Qivr5 and Qivr19. In addition, we observed several regions of the genome with suggestive linkage. There are potentially complex combinatorial interactions of the parental alleles among loci. Analysis of multiple gene expression data sets and sequence variants in these strains highlights about 30 strong candidate genes across all loci that may control influenza A susceptibility and resistance. CONCLUSIONS: We have mapped influenza susceptibility loci to chromosomes 2, 5, 16, 17, and 19. Body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. We highlight candidate genes in the respective intervals and review their possible biological function during infection.     

Integrating QTL and high-density SNP analyses in mice to identify Insig2 as a susceptibility gene for plasma cholesterol levels

The use of inbred strains of mice to dissect the genetic complexity of common diseases offers a viable alternative to human studies, given the control over experimental parameters that can be exercised. Central to efforts to map susceptibility loci for common diseases in mice is a comprehensive map of DNA variation among the common inbred strains of mice. Here we present one of the most comprehensive high-density, single nucleotide polymorphism (SNP) maps of mice constructed to date. This map consists of 10,350 SNPs genotyped in 62 strains of inbred mice. We demonstrate the utility of these data via a novel integrative genomics approach to mapping susceptibility loci for complex traits. By integrating in silico quantitative trait locus (QTL) mapping with progressive QTL mapping strategies in segregating mouse populations that leverage large-scale mapping of the genetic determinants of gene expression traits, we not only facilitate identification of candidate quantitative trait genes, but also protect against spurious associations that can arise in genetic association studies due to allelic association among unlinked markers. Application of this approach to our high-density SNP map and two previously described F2 crosses between strains C57BL/6J (B6) and DBA/2J and between B6 ApoE(-/-) and C3H/HeJ ApoE(-/-) results in the identification of Insig2 as a strong candidate susceptibility gene for total plasma cholesterol levels.     

Quantitative trait loci for body weight in the intercross between SM/J and A/J mice.

We performed a genome-wide quantitative trait locus (QTL) analysis of body weight at 10 weeks of age in a population of 321 intercross offspring from SM/J and A/J mice, progenitor strains of SMXA recombinant inbred strains. Interval mapping revealed two significant QTLs, Bwq3 (body weight QTL3) and Bwq4, on Chromosomes (Chrs) 8 and 18 respectively, and five suggestive QTLs on Chrs 2, 6, 7, 15 and 19. Bwq3 and Bwq4 explained 6% of the phenotypic variance. The SM/J alleles at both QTLs increased body weight, though the SM/J mouse was smaller than the A/J mouse. On the other hand, four of the five suggestive QTLs detected had male-specific effects on body weight and the remainder was female-specific. These suggestive QTLs explained 5-6% of the phenotypic variance and all the SM/J alleles decreased body weight.     

γ-Glutamyl Cyclotransferase: A New Genetic Polymorphism in the Mouse (MUS MUSCULUS) Linked to LYT–2

Electrophoretically variant forms of gamma-glutamyl cyclotransferase have been identified in red cells of inbred mouse strains. Each inbred strain exhibited a major band of activity and a minor band that migrated more anodally. The polymorphism affects the migration of both the major and minor bands in a similar way. F1 hybrids between strains with fast forms (A/J) and strains with the slow forms (C57BL/6J) exhibited a four-banded pattern consistent with co-dominant inheritance. The patterns observed in backcross and F2 mice were consistent with the segregation of a pair of autosomal co-dominant alleles. Recombinant inbred strains and a congenic strain were used to show that the locus controlling gamma-glutamyl cyclotransferase (Ggc) is linked to Lyt-2, a lymphocyte alloantigen locus on chromosome 6, with an estimated map distance of 5.0 +/- 2.5 centimorgans.     

Using progenitor strain information to identify quantitative trait nucleotides in outbred mice

We have developed a fast and economical strategy for dissecting the genetic architecture of quantitative trait loci at a molecular level. The method uses two pieces of information: mapping data from crosses that involve more than two inbred strains and sequence variants in the progenitor strains within the interval containing a quantitative trait locus (QTL). By testing whether the strain distribution pattern in the progenitor strains is consistent with the observed genetic effect of the QTL we can assign a probability that any sequence variant is a quantitative trait nucleotide (QTN). It is not necessary to genotype the animals except at a skeleton of markers; the genotypes at all other polymorphisms are estimated by a multipoint analysis. We apply the method to a 4.8-Mb region on mouse chromosome 1 that contains a QTL influencing anxiety segregating in a heterogeneous stock and show that, under the assumption that a single QTN is present and lies in a region conserved between the human and mouse genomes, it is possible to reduce the number of variants likely to be the quantitative trait nucleotide from many thousands to <20.     

Towards construction of a high resolution map of the mouse genome using PCR-analysed microsatellites.

Fifty sequences from the mouse genome database containing simple sequence repeats or microsatellites have been analysed for size variation using the polymerase chain reaction and gel electrophoresis. 88% of the sequences, most of which contain the dinucleotide repeat, CA/GT, showed size variations between different inbred strains of mice and the wild mouse, Mus spretus. 62% of sequences had 3 or more alleles. GA/CT and AT/TA-containing sequences were also variable. About half of these size variants were detectable by agarose gel electrophoresis. This simple approach is extremely useful in linkage and genome mapping studies and will facilitate construction of high resolution maps of both the mouse and human genomes.     

Mononucleotide repeats are an abundant source of length variants in mouse genomic DNA

Microsatellite sequences, such as dinucleotide repeats, show a high degree of polymorphism in eukaryotic DNA. These sequences are convenient as genetic markers and can be analyzed by the polymerase chain reaction (PCR). We have assessed the frequency of length variants in 18 mononucleotide repeats in mouse DNA and find that the variability is similar to that reported for dinucleotide repeats. Nine of the 18 repeat sequences (50%) have three or more alleles in the strains tested. Ten of these repeat sequences have been mapped using strain distribution patterns (SDPs) in recombinant inbred (RI) strains.     

辽宁省6种常用近交系小鼠10个微卫星位点的遗传质量检测报告

目的利用微卫星技术对辽宁省6种近交系小鼠进行遗传质量分析。方法根据Mouse Genome Database和相关文献选取10个多态信息丰富的位点和引物,进行PCR扩增和PAGE电泳,对小鼠的遗传多态性进行研究。结果不同品系小鼠同一位点的扩增结果表现出多态性,同一品系同一位点表现单态性,所有小鼠的10个位点都处于纯合状态;遗传距离分析表明,C57BL/10与C57BL/6J小鼠之间的遗传距离最近,为0.1021,遗传距离最远的是BALB/c与C57BL/10、C57BL/6J,分别为0.1635和0.1614。结论运用所筛选的10个微卫星位点可以对近交系小鼠进行遗传质量检测,说明该方法具备可行性。     

Polymorphism for PCR-analyzed microsatellites between the inbred mouse strains LG and SM

Using agarose gel electrophoresis, we surveyed four strains of inbred mice (AKR/J, C57BL/J, LG/J, and SM/J) for 472 microsatellite loci. Agarose electrophoresis proved to be extremely efficient in separating alleles differing by six or more base pairs and detected a majority of allelic differences of between two and six base pairs. Overall, 64.4% of loci showed polymorphism among the four strains, and pairwise comparisons ranged from 42.1% to 48.1%. Microsatellite polymorphism for strains LG/J and SM/J has not been previously described and was sufficiently high (47.1%) to make these size-divergent strains excellent candidates for quantitative trait loci (QTL) analysis of normal growth.     

Unique genetic variation revealed by a microsatellite polymorphism survey in ten wild-derived inbred strains

Here we report on a genome polymorphism survey using 254 microsatellite markers in ten recently wild-derived inbred strains. Allele size analysis showed that the rate of polymorphism of these wild-derived mouse strains when compared with any of the common laboratory strains is on average 79.8%. We found 632 wild-derived alleles that were not present in the common laboratory strains, representing a 61% increase over the genetic variation observed in the laboratory strains. We also found that on average 14.5% of the microsatellite alleles of any given wild-derived inbred strain were unique. Our results indicate that the recently wild-derived mouse strains represent repositories of unique naturally occurring genetic variability and may prove invaluable for the study of complex phenotypes and in the construction of new mouse models of human disease.