Chemical mutagenesis of the mouse genome: an overview

The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a gene driven approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a phenotype driven approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.     

Molecular Characterization of the pun Allele of the Mouse Pink-Eyed Dilution Locus

The mouse pink eyed dilution locus, p, located on chromosome 7, mediates coat and eye color. The human correlate of this gene may underlie some forms of tyrosinase-positive oculocutaneous albinism. Mutations at the p locus result in a reduction in pigmentation of the eyes and coat. Although most mutant p alleles (including all spontaneous mutations) affect only pigmentation, several mutant alleles (all radiation induced) are also associated with a variety of other phenotypes. We have focused our attention on the p^un mutant allele, a spontaneous mutation, exhibiting one of the highest reversion frequencies reported for a mammalian mutation. Using a new technique, genome scanning, we have cloned fragments of genomic DNA from the p locus that are associated with a DNA duplication in p^un DNA. These fragments can now be used to locate the p gene-encoding sequences and aid in the molecular characterization of complex mutant p alleles.     

长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法的建立及初步应用

目的建立长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物MHV的检测。方法根据已发表的小鼠肝炎病毒（MHV）S基因序列,设计合成引物。提取MHV细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、稳定性、重复性试验。并对65只长爪沙鼠及12只小鼠进行检测。结果建立的MHVRT-PCR检测方法特异、敏感、稳定。以MHVRNA逆转录产物为模板,所能检测RNA最小模板浓度为3．1pg／μL,可检测病毒最小滴度为10^-3／mL。65只沙鼠经RT-PCR检测,均为阴性,12只小鼠经RT．PCR检测,有3只MHV阳性,测序结果与Genbank中MHV核酸序列同源性均为97％。结论建立的长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法可用于长爪沙鼠、小鼠等实验动物MHV的检测。     

Nosema algerae: Infection of the white mouse by a mosquito parasite

Spores of Nosema algerae, a microsporidan parasite of mosquitoes, were subcutaneously injected into the ears, tail, and feet of white mice. Infections were transient and localized at the injection sites. Spore germination tests in blood plasma indicated that it is unlikely that spores injected by an infected mosquito bite would result in an infection.     

Morphological and functional changes in the tight junctions of the bile canaliculi induced by bile duct ligation

Summary Thin sections after bile duct ligation showed that the depth of tight junctions appeared to increase and that the distance between individual punctate contacts appeared to become irregular and wider than in controls. The freeze fracture replicas clearly demonstrated these changes in the tight junction morphology. Changes were noted most conspicuously in the tight junction three weeks after ligation. Measurements of the junctional morphology in control and ligated specimens showed that the junctional depth had increased two fold in the latter, whereas the number of strands had scarcely changed. Lanthanum tracer experiments showed that the tight junctions did not permit the passage of the tracer in normal nor ligated rats. It was concluded that the mechanism of obstructive jaundice could not be related to changes in junctional morphology causing increased junctional permeability.Tight junction depth in this paper is synonymously used with Tight junction width or Tight junction thickness     

遗传性BALB／c白内障小鼠模型培育初报

在生产实践中发现了两只自然突变的白内障雄小鼠,后与BALB／c和C3H／HEJ雌鼠交配,它们的后代雌雄均有白内障个体出现,提示这是一个常染色体显性基因。用传统的方法与BALB／c回交,试图把白内障基因导入BALB／c品系中．旨在建寺一个溃传性BALB／ccat/Cat白内障小鼠模犁。     

小鼠常压急性缺氧模型装置及其应用

设计了一种建立小鼠常压急性缺氧模型的简易装置,并通过实验与常规缺氧模型装置进行了比较。结果表明：此装置避免了高浓度C02或低气压对实验的影响,而且造价低廉,制作简单,使用方便,为完善缺氧的研究提供了一种手段。     

无细胞百日咳菌苗毒性试验参考苗制备条件的探讨

将不同灭活条件下制备的无细胞百日咳毒性试验参考苗进行了检测。结果表明,以浓度０．１％Ｆｏｒｍａｌｉｎ溶液２５℃灭活９６－１２０小时制备的无细胞百日咳菌苗毒性试验参考苗,凝集效价仍可达到百日咳Ⅰ相血清原效价;不耐热毒素试验呈阴性;毒性试验ＢＷＤＵ／ｍｌ为７５．９－１２８．４;ＬＰＵ／ｍｌ为２．１－６．０;ＨＳＵ／ｍｌ为３．９－５．７;稳定性良好。该苗可标化作为无细胞百日咳菌苗毒性试验的参考标准     

Comparison of fecal biota from specific pathogen free and feral mice

Specific pathogen free (SPF) rodents are derived from germfree animals that are colonized with Schaedler's flora, a cocktail of eight bacterial strains isolated from the natural biota of mice. During successive generations SPF animals acquire a complex biota, but it is not known how similar it is to natural mouse biota. Therefore, fecal pellets of two feral mice and three SPF mice were studied by small subunit ribosomal DNA sequence analysis. After amplification of 16S rDNA by Bacterial Kingdom-specific primers, 132 rDNA clones from feral mice and 219 clones from SPF mice were placed phylogenetically. Forty-four percent of recovered rDNAs from feral mice were from organisms belonging to the Ribosomal Database Project's Bacteroides Group with significant proportions also coming from lactobacilli, the Clostridium coccoides Group and the Clostridium leptum Group. Although the SPF biota appeared equally complex at lower phylogenetic levels, the major phylogenetic groups represented were less diverse in that 92% of rDNA's from SPF mice mapped to groups of clostridia with 79% to the C. coccoides Group alone. Given the number of physiological parameters influenced by the gut biota and the importance of mice in biomedical research, further investigations are warranted.     

Mouse Forward Genetics in the Study of the Peripheral Nervous System and Human Peripheral Neuropathy

Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics. Special issue article in honor of Dr. George DeVries.     

Substructure of solitary cilia in mouse kidney

Summary Recent scanning electron microscopic studies confirm the presence of solitary cilia on most epithelial cells along the mammalian nephron and collecting ducts.By transmission electron microscopy we have found that the axonemata of such cilia consist of a maximal number of 9 doublet and no singlet filaments. 10% of the cross-sectioned cilia contain 9 doublets arranged in a peripheral ring (9+0 pattern). 30 % of the cross-sections contain 8 or 7 doublets in peripheral ring and 1 or 2 doublets in the central region (8+1 and 7+2 patterns). Serial sections and goniometer tilt reveal the central doublets to originate as dislodged peripheral doublets. 60% of the sectioned cilia contain filament numbers between 8 and 4. In patterns of 5 and 4 filaments single microtubules predominate.The functional significance of these atypical cilia is discussed.We are indebted to Prof. B. Afzelius and Prof. Th. Brun for valuable information and discussions during this work. The technical assistance of Miss K. Weltzin, Mr. E. Erichsen, Mr. R. Jensen and Mr. J. Røli is greatly appreciated     

Characterization of the antiviral activity constitutively produced by murine conceptuses: absence of placental mRNAs for interferon alpha and beta

Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species.     

实验小鼠在癌症研究中的应用及其进展

小鼠是生物医学研究中使用数量最多的哺乳类实验动物。人类利用小鼠模型进行癌症研究已有100多年的历史,小鼠大量的遗传变异可作为研究人类癌症的借鉴。特别是近年来,培育成功的转基因、基因敲除等遗传工程小鼠模型,使我们对人类癌症发生有了深刻的认识,为评估癌症的诊断方法,革新预防和治疗方案提供了一个很有价值的平台。本文着重介绍了癌症研究中常用的小鼠模型、GEM模型及取得的最新进展等,分析了小鼠肿瘤模型的局限性,并对其发展趋势进行展望。     

小鼠39个微卫星的PCR条件及其运用

目的探索小鼠基因组39个微卫星的PCR条件,评价微卫星在小鼠遗传检测中的运用.方法采用梯度法探索39个微卫星的PCR条件;选择本中心不同来源及引种时间的C57BL/6、BALB/c、DBA/2J、CBA/N、FVB/NJ、ICR共6个品系(8个组)小鼠,每组采用10只个体的鼠尾,提取DNA并混合成DNA池,用39个微卫星扩增后电泳观察、比较种系纯度.结果小鼠微卫星的PCR条件差异较大,Mg2+浓度多数在1.5 mmol/L左右,退火温度多数在59℃左右.在6个品系小鼠的39个微卫星位点中,C57BL/6、BALB/c、DBA/2J都是纯合的; 其余品系有1～3个杂合位点. BALB/c在D5Mitl68、D8Mit320、D13Mit262三个位点,DBA/2J在D14Mit205位点与数据库记录有差异.结论本研究为小鼠39个微卫星提供了候选的PCR条件,并对6个品系小鼠的微卫星概貌及微卫星的运用价值进行了探讨.     

小鼠睾丸表皮生长因子受体及增殖细胞核抗原表达的加龄变化研究

观察了表皮生长因子受体及增殖细胞核抗原在生后１天至生后１０月龄昆明种小鼠睾丸内的表达,结果表明：精原细胞及初级精线产细胞从生后第２周至生后４周龄ＤＮＡ复制旺盛,增殖细胞核抗原免疫反应阳性细胞面密度于生后１４天出现峰值。生长因子受体在间质细胞、精母细胞内均有表达。生后４周时,精母细胞表皮生长因子受体表达较强,便于表皮生长因子发挥调节细胞增殖、调亡的作用。     

Refinement of the mouse model of congenital toxoplasmosis

The goals of the present investigation, focusing on the BALB/c mouse model of congenital toxoplasmosis, were: (1) to find a method to determine pregnancy in the mouse. The method has 100% sensitivity and 72% specificity; (2) to test congenital transmission during the chronic stage of toxoplasmosis. This occurred in 2 of 10 mice tested; (3) to investigate the relationship between the infective dose and the rate of congenital transmission. This was not demonstrated for doses of 10(2) to 10(3) bradyzoites and oocysts of Prugniaud, M3 and M7741 strains, with transmission rates of 3 of 8 to 6 of 10 mice inoculated; (4) to determine homologous and heterologous protection. Homologous protection was demonstrated with Prugniaud cysts, and heterologous protection was found between ME-49 and M3 cysts. This finding is consistent with the uniform natural protection against congenital toxoplasmosis seen in immune women and ewes.     

Proteolysis of Mutant Huntingtin Produces an Exon 1 Fragment That Accumulates as an Aggregated Protein in Neuronal Nuclei in Huntington Disease

Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.     

蛋白质粉对小鼠免疫功能的影响

目的探讨蛋白质粉对正常小鼠免疫调节作用。方法将BALB/c小鼠随机分为3批,每批分为4组,分别进行了小鼠免疫器官/体质量比值测定和小鼠碳廓清实验;绵羊红细胞诱导小鼠DTH、抗体生成细胞检测和血清凝血素测定（HC50）;ConA诱导的小鼠脾淋巴细胞转化实验和乳酸锂脱氢酶法（LDH）测定NK细胞活性;小鼠腹腔巨噬细胞吞噬鸡红细胞实验。结果10.00 g/kg剂量的蛋白质粉可增强绵羊红细胞诱导小鼠DTH能力（P〈0.05）,促进抗体生成细胞数的生成（P〈0.01）。3.33 g/kg和10.00 g/kg剂量组能促进ConA诱导的小鼠脾淋巴细胞转化能力（P〈0.05或P〈0.01）和血清凝血素的生成（P〈0.05）;三个剂量组均能提高小鼠腹腔巨噬细胞吞噬鸡红细胞能力（P〈0.05或P〈0.01）;3.33 g/kg和10.00 g/kg剂量组能提高NK细胞活性（P〈0.05）;但对小鼠碳廓清能力和免疫器官/体重比值无明显影响。结论蛋白质粉对正常小鼠的细胞、体液免疫和单核-巨噬细胞功能和NK功能有促进作用,即具有增强免疫力功能。     

不同年龄小白鼠全脑细胞质氨酰tRNA合成酶活力的比较

从不同年龄(20天,30天,1年)的小白鼠全脑制得细胞质混合氨酰tRNA合成酶。用异源体系(即用酵母tRNA和小白鼠全脑氨酰tRNA合成酶)测定了氨酰tRNA合成酶分别载运~3H标记的Asp、Gly、Glu、Lys和Ala的活力。结果表明除未检出tRNA~(Glu)的合成酶活力外,对其余四种氨基酸都有明显的活力,特别是年龄20天小白鼠的氨酰tRNA合成酶对~3H-Gly具有高达35％的载运活力。对~3H-Gly、~3H-Lys和~3H-Ala的载运活力有随增龄而下降的趋势,但对~3H-Asp的载运活力则随年龄增长而增高。