近交系小鼠微卫星DNA引物的筛选和Tm值优化研究

目的通过对近交系小鼠微卫星引物的筛选和Tm值优化研究,以探索小鼠DNA多态性检测方法。方法随机选用32对位于小鼠不同染色体的微卫星引物,用PCR扩增方法对常用C57BL/6、C3H、BALB/c、DBA/2、129、FVB及SCID近交系小鼠DNA多态性进行扩增和电泳分析,并对其中25对引物的Tm值进行优化。结果 25对引物可稳定扩增,2对引物在不同品系小鼠间表现为单态性,23对引物在不同品系间呈多态性,10对引物呈显著多态性(3～4个态性)。结论所筛选和优化的25对微卫星引物,对不同品系小鼠DNA可稳定扩增,其电泳结果具有较高的DNA多态性,可反映不同品系小鼠的遗传概貌。     

常用近交系小鼠微卫星DNA多态性的分析研究

摘要：目的用微卫星引物对常用近交系小鼠进行遗传监测和DNA多态性分析。方法通过Genome Database选 取位于小鼠不同染色体上的15个微卫星位点,应用PCR扩增技术对近交系小鼠C57BL／J、DBA／2、C3H、BALB／e、 129、FVB、SCID进行微卫星DNA多态性的分析。结果15个微卫星位点除D8Mitll3、D13Mit88无扩增条带外,其 余13个微卫星位点引物对七种近交系小鼠均有稳定扩增,同一品系不同个体之间表现为单态性;不同品系个体之 间表现为多态性。结论所用微卫星位点具有信息含量高、分布广、扩增稳定、高度多态性的特点,各位点PCR的 稳定扩增,依其DNA多态性的特点来判定各品系间的遗传差异,是一种准确客观、方便快速的遗传监测方法。     

Genetic defect in secretion of complement C5 in mice.

A genetic deficiency of the fifth (C5) component of complement1-3, a serum glycoprotein of molecular weight (MW) 220,000 (ref. 4), has been found in 39% of inbred strains of mice3. Sera of deficient mice lack detectable C5 activity and protein2,3. In addition deficient mice produce antibody to mouse C5 when injected with sera from C5 sufficient (normal) strains. Levy et al.5 showed that somatic cell hybrids between C5 deficient (B10.D2/old line) macrophages and either C5 sufficient (B10.D2/new line) mouse kidney or chicken erythroblasts secreted haemolytically active mouse C5 in vitro. Several possible molecular mechanisms to account for the findings were considered, but insufficient direct data were available to choose among them. We recently reported that mouse (CD.1 strain) peritoneal cells in culture synthesise and secrete a single chain precursor, pro-C5 (MW approximately 210,000), of the two-chain (alpha chain, 125,000 and beta chain 83,000 MW) C5 protein6. Radiolabelled precursor C5 was contained within the cells and was secreted into the tissue culture media. Using similar methods, we now find that C5 deficiency in each of five different mouse strains (AKR, SWR, DBA/2J8 A/HeJ and B10.D2/old line) is due to a failure in secretion of C5 protein and not to a failure in biosynthesis of pro-C5.     

三种小鼠血液生理生化正常值的测定

本文通过对三种小鼠－ＢＡＬＢ／ｃ、Ｃ５７ＢＬ／６Ｊ近交系小鼠和昆明远交群小鼠的血液学和血液生化指标进行测定。其结果：⑴血液学指标：ＢＡＬＢ／ｃ近交系小鼠的嗜中性白细胞、淋巴细胞雌雄间差异显著;Ｃ５７ＢＬ／６Ｊ近交系小鼠、昆明小鼠雌雄间差异不显著;昆明小鼠的白细胞比ＢＡＬＢ／ｃ、Ｃ５７ＢＬ／６Ｊ近交系小鼠的低;ＢＡＬＢ／ｃ近交系小鼠的单核细胞、嗜酸性白细胞、嗜碱性白细胞记数比Ｃ５７ＢＬ／６Ｊ近交系小     

Mapping of genetic modifiers of <Emphasis Type="Italic">Plcd1</Emphasis> in scant hair mice (<Emphasis Type="Italic">snthr</Emphasis><Superscript><Emphasis Type="Italic">1Bao</Emphasis></Superscript>)

The scant hair mutant mouse (locus symbol: snthr ^1Bao ) is a recessive mutation that originated in an ethylnitrosourea chemical carcinogenesis study using the DBA/2J inbred strain. The gene responsible for the mutation was previously determined to be phospholipase C, delta 1 (Plcd1; mutant allele symbol Plcd1 ^snthr1Bao ). To map the modifiers of Plcd1, an intercross (DBA/2J-snthr ^1Bao /snthr ^1Bao × C57BL/6J+/+) was conducted. The F2 mutant progeny exhibited a variety of alopecia phenotypes; all F2 mutants (n=507) were classified into 3 groups (mild, moderate, and severe alopecia) and genotyped based on 96 microsatellites. A major QTL was identified on mouse chromosome (mChr) 15 at 12 cM with an LOD score greater than 7 (P < 0.0001). Three minor QTLs were detected on mChr 2, 5, and 7 at 40, 84 and 48 cM, respectively. The QTLs on mChr 7 and 15 were associated with minor alopecia while the QTLs on mChr 2 and 5 were associated with moderate to severe alopecia. No antagonistic or synergistic effects among or between the 4 QTLs were found. Integrating the functions of the 4 potential regulatory QTLs and mutant Plcd1 ^snthr1Bao , we found that these QTLs might contribute to variations of scant hair severity by altering the Ca²⁺ signal pathways in mouse skin.     

Dissecting anxiety-related QTLs in mice by univariate and multivariate mapping

To detect genes underlying anxiety-related traits in mice,we performed univariate and multivariate QTL mapping analyses of phenotypes obtained from 71 mice of the BXD recombinant inbred (RI) strains (n=528 mice) and their parental strains (C57BL/6J and DBA/2J).Separate and joint mapping analyses were carried out using a linkage map composed of 506 simple sequence repeats (SSRs).The main QTL effects,interactions between pairs of QTLs (epistasis),and their environmental interactions were estimated.The results showed that anxiety-related traits were influenced by multiple QTLs (five main effect QTLs and three epistatic QTLs).Ten potential anxiety-related candidate genes within the QTL intervals on chromosomes 5,13 and 15 were identified.Some of these genes have been reported previously to be associated with the anxiety response.Based on our results,it is suggested that the multivariate QTL mapping approach improves the statistical power for detecting QTL and the precision of parameter estimation.Moreover,multivariate mapping can also detect pleiotropic QTL effects.     

Amplification of immunoglobulin lambda constant genes in populations of wild mice

The lambda immunoglobulin light chain (Ig lambda) locus of BALB/c inbred mice consists of two variable region gene segments (V lambda)1-3, and four constant region gene segments (C lambda)1,2,4,5. Each C lambda gene segment is associated with a unique joining segment (J lambda)2,4-7, and they are organized in two paired units, J3C3-J1C1 and J2C2-J4C4 (refs 4, 8). Using cDNA probes specific for C lambda 1 and C lambda 2 (ref. 9) we have analysed the genomic organization of the C lambda gene segments in wild-derived and inbred strains of mice. Although Southern blots of the genomic DNA of inbred mice show a constant pattern of hybridization, wild-derived mice show a high degree of variation in the number, size and intensity of hybridizing fragments. We have now found that, per haploid genome, mice of a Mus musculus musculus stock isolated from Sladeckovce, Czechoslovakia (CzII) have at least 12 C lambda segments, and mice of a Mus musculus domesticus stock 'Centreville Lights' from Centreville, Maryland (CL) have at least 8 C lambda segments. There appears to have been relatively recent amplifications of the C lambda gene segments in wild mice.     

The mosaic structure of variation in the laboratory mouse genome

Most inbred laboratory mouse strains are known to have originated from a mixed but limited founder population in a few laboratories. However, the effect of this breeding history on patterns of genetic variation among these strains and the implications for their use are not well understood. Here we present an analysis of the fine structure of variation in the mouse genome, using single nucleotide polymorphisms (SNPs). When the recently assembled genome sequence from the C57BL/6J strain is aligned with sample sequence from other strains, we observe long segments of either extremely high (approximately 40 SNPs per 10 kb) or extremely low (approximately 0.5 SNPs per 10 kb) polymorphism rates. In all strain-to-strain comparisons examined, only one-third of the genome falls into long regions (averaging >1 Mb) of a high SNP rate, consistent with estimated divergence rates between Mus musculus domesticus and either M. m. musculus or M. m. castaneus. These data suggest that the genomes of these inbred strains are mosaics with the vast majority of segments derived from domesticus and musculus sources. These observations have important implications for the design and interpretation of positional cloning experiments.     

用于乙肝疫苗效力检定的小鼠种群的建立

目的在进行乙肝疫苗效力检测时应用统一的动物种群。方法目前评价乙肝疫苗免疫效力的主要方法是用小鼠测定乙肝疫苗的ED50值(接种疫苗后使50%的动物抗体阳转的疫苗稀释度的倒数),而小鼠对HBsAg免疫应答的水平与H-2单倍型密切相关。因此我们通过微量细胞毒法和PCR方法检测小鼠的H-2单倍型的特点,得出乙肝疫苗与不同动物种群的相关性,解决实际工作中的问题。结果通过多方面的实验验证,NIH鼠可以对乙肝疫苗产生高应答效应。结论NIH鼠的H-2单倍型中有q型基因,同时NIH鼠保持了原封闭群动物繁殖力强生长速度快等优点,所以便于进行乙肝疫苗效力检测时应用。     

利用优选的微卫星标记建立常用大小鼠品系的遗传监测体系

摘要 目的 利用微卫星位点在不同近交系小鼠、大鼠中可能具有的多态性,分别筛选出可以一次性区别5种常用近交系小鼠和3种常用近交系大鼠的微卫星引物组合,为近交系小鼠和大鼠的遗传监测分析提供高效的微卫星位点与简易可靠的鉴定条件。 方法 通过查询相关数据库以及文献报道,按照已有明确的微卫星实验数据记录为原则,进行比对和筛选,组成了小鼠37 个和大鼠24个微卫星位点库。 通过PCR扩增和5%琼脂糖电泳鉴定,优化筛选出可以区分单一品系和所有品系的微卫星位点组合。 结果 小鼠优选出11个位点可用于5种常用近交系的鉴定,并建立了1个包含6个微卫星位点的库可用于5种小鼠品系的遗传监测;大鼠优选出6个微卫星位点,可用于3种常用近交系的监测。 结论 成功建立了一套可以区分常用5种小鼠和3种大鼠品系的微卫星监测方法。     

C57BL/6J胚胎冷冻小鼠的遗传监测和分析研究

目的用生化标记和微卫星检测法观察近交系C57BL/6J小鼠胚胎冷冻后的遗传质量是否发生改变。方法采用国标(GB/T 14927.1-2008)生化标记基因检测法对4只玻璃化冷冻、解冻移植后获得的C57BL/6J F1代小鼠的13个生化位点进行预检测,再根据微卫星DNA多态性的遗传监测方法进行多态性分析。结果4只玻璃化冷冻后移植获得的C57BL/6 F1代小鼠的遗传概貌与国标(GB/T 14927.1-2008)中的14个生化位点完全一致;并与本文作者已建立"几种常用近交系小鼠微卫星DNA多态性的分析研究"的遗传监测结果完全相符。结论玻璃化冷冻法(EFS40,straw)对C57BL/6J冷冻胚胎小鼠的遗传物质并未造成影响,其遗传特性保持稳定,是可行的小鼠胚胎冷冻方法。     

H-2基因检测方法在北京市实验动物遗传质量抽检中的应用

目的为贯彻实施新国标,通过检测小鼠H-2单倍型,确认近交系小鼠免疫遗传质量。方法依据新国标GB/T14927.2-2008中的微量细胞毒检测方法,检测北京地区主要生产厂家的BALB/c和C57BL/6小鼠共70只的H-2单倍型。结果共抽检4个单位的BALB/c和C57BL/6近交系小鼠70只,经检测BALB/c为H-2Dd和H-2Kd,C57BL/6为H-2Db和H-2Kb,符合率100%,质量符合要求。结论经抽检,北京地区主要生产厂家2个品系小鼠免疫遗传质量合格。     

High frequency of unequal recombination in pseudoautosomal region shown by proviral insertion in transgenic mouse

The mammalian X and Y chromosomes, in contrast to the autosomes, pair during male meiosis only near the telomeres. Alleles localized in this region can undergo reciprocal exchange during meiosis. Because such sequences do not show strict sex-linked inheritance, they have been termed pseudoautosomal. In man, several DNA sequences have been described which show pseudoautosomal transmission and which are localized in the pairing region at the ends of the short arms of both the X and Y chromosomes (refs 6-9, and D. Page, unpublished results). We now show that the transgenic mouse strain, Mov-15, contains a single Moloney murine leukaemia virus (M-MuLV) genome in its germline, and genetic evidence indicates that the provirus is integrated into the pseudoautosomal region of the sex chromosome. Proviral copies are lost or gained in 7% of male meioses in this strain, and mouse sequences flanking the provirus are tandemly repeated and highly variable. We conclude that unequal recombination events occur with high frequency in the pairing region, possibly because of the presence of repeated sequences.     

Retrovirus-induced de novo methylation of flanking host sequences correlates with gene inactivity

The pattern of DNA methylation changes during development of eukaryotes, and hypomethylation frequently correlates with gene expression (for reviews see refs 1-4). A causal relationship between hypermethylation and gene inactivity has been established for retroviral genomes which are methylated de novo when inserted into the germ line of mice (ref. 5; for review, see ref. 6). The mutual interaction of the provirus with the host genome can influence virus expression and can result in inactivation of the host gene by insertional mutagenesis. We report here that the insertion of a provirus can change the methylation pattern of the host DNA. Sequences flanking the provirus become methylated de novo within 1 kilobase (kb) of the integration site. In Mov-13 mice, which carry a lethal mutation of the alpha 1(I) collagen gene, de novo methylation of host DNA is associated with a change in chromatin conformation. This suggests that virus-induced DNA methylation can alter DNA-protein interactions and thereby interfere with correct gene activation during embryonic development.     

Paternal inheritance of mitochondrial DNA in mice.

For nearly 20 years it has been assumed on the basis of low-resolution experiments that mitochondrial (mt)DNA, in contrast to the genes in the nucleus, has an exclusively maternal mode of inheritance in animals. Using the polymerase chain reaction, paternally inherited mtDNA molecules have now been detected in mice at a frequency of 10(-4), relative to the maternal contributions. These mice were hybrids between two inbred strains (C57BL/6J and Mus spretus) whose mtDNAs can be distinguished easily. This new mode of inheritance provides a mechanism for generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission.     

Endogenous mammary tumour virus DNA varies among wild mice and segregates during inbreeding.

Proviruses of the mouse mammary tumour virus (MMTV) endogenous to normal mice can be identified by molecular hybridisation and distinguished using restriction endonucleases. Feral mice display marked heterogeneity with respect to the number of copies and the sites of insertion of endogenous MMTV-specific DNA, with occasional mice apparently free of MMTV DNA. Several different MMTV proviruses present in laboratory mice have segregated like stable, independent genetic elements during the inbreeding which followed a cross between Bagg albino and DBA mice 60 years ago. The results favour the hypothesis that endogenous proviruses have been established by multiple, independent infections of germ cells rather than by somatic mutation of ancestral proviruses or of cellular genes.     

Investigation of genetic linkage between myosin and actin genes using an interspecific mouse back-cross

The introduction of cloned probes to follow the segregation of DNA restriction fragment length polymorphisms (RFLPs) has led to a revival of mendelian genetics in attempts to map the human genome. In the mouse, however, it has often proved difficult to detect an RFLP with a DNA probe between different inbred strains of the laboratory mouse. To circumvent this problem, we have used two species, Mus musculus domesticus and Mus spretus which interact as sympatric species but can be interbred under laboratory conditions. Because of the relative evolutionary distance between these species, they exhibit polymorphism at many more loci than do different strains of the usual M. m. domesticus laboratory mouse. This is also observed at the DNA level when the sizes of restriction fragments encoding a specific gene are compared. We have used these RFLPs between M. m. domesticus and M. spretus to follow the segregation of genes encoding different isoforms of myosin alkali light chains in the backcross progeny between these species and to compare this with that of other contractile protein genes. No linkage between these genes was observed.     

BALB/c小鼠遗传污染对单克隆抗体制备的影响

目的检测BALB/c小鼠遗传污染对单克隆抗体制备实验的影响。加强近交系小鼠的饲养管理和重视实验动物的遗传质量检测。方法使用遗传生化标记方法对购进的两批BALB/c小鼠进行了碱性磷酸酶-1(Akp-1)等13个生化位点的检测。结果注射杂交瘤细胞后不产生腹水,经生化检测发现,购买的第一批BALB/c小鼠,在过氧化氢酶-2(Ce-2)等4个生化位点发现杂合基因,第二批BALB/c小鼠在过氧化氢酶-2(Ce-2)等8个生化位点发现杂合基因。结论本实验说明在实验动物的饲养管理上存在漏洞,上述两批BALB/c小鼠未严格按照国家实验动物管理操作规程操作,必须规范实验动物饲养管理,加强实验动物遗传检测,这是保证动物试验结果准确可靠不可或缺的重要环节。     

53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2(fl/-)) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.