Uncv无毛小鼠无毛基因的分子克隆及序列分析

目的 探讨Unev无毛小鼠的无毛性状与无毛基因(hairless gene,hr)的相关性.方法 参照Gen-Bank上公布的小鼠的hr序列,设计5对引物,用RT-PCR方法对本单位培育的Unev无毛小鼠hr的编码区序列进行了克隆与分析.结果 获得了Unev无毛小鼠及野生型hr的全部编码区序列(3546 bp).Unev无毛小鼠hr基因与野生型小鼠hr基因的长度及序列完全一致,同源性为100%.与GenBank上发表的国外小鼠hr基因序列(Z32675)相比,同源性为99.7%,共10个碱基发生了突变,其中2个碱基突变导致了相应的氨基酸突变;和昆明小鼠的hr(AY547391)相比,同源性为99.6%,共12个碱基发生了突变,其中3个碱基突变导致了相应的氨基酸突变;但这些突变是由种属差异造成的.结论 Uncv无毛小鼠的无毛性状产生与hr基因无关.     

使用超净工作台实施子宫摘除术净化KM小鼠的研究

目的建立SPF级湖北封闭群KM小鼠生产群.方法使用超净工作台实施子宫摘除术净化小鼠的方法,建立了SPF级湖北封闭群KM小鼠生产群,并对子一代进行了微生物及寄生虫的检测.结果使用上述方法实施手术67例,成功57例,得净化仔鼠509只,育成的460只小鼠符合SPF级标准.结论使用生物净化的方法可以保存优良实验动物品种.     

SPF级小鼠超数排卵效果

目的探求小鼠周龄、品系和季节对SPF级小鼠超数排卵效果的影响。方法在春、夏、冬季对4～10周龄的SPF级BALB／c小鼠和KM小鼠进行超数排卵处理,通过见栓率、产胚率、3．5d胚胎获得量及胚胎类型等方面进行统计学分析。结果小鼠周龄、品系和季节对SPF级BALB／c小鼠和KM小鼠见栓率没有影响;对产胚率、3．5d胚胎获得量和胚胎类型等方面有一定影响。结论SPF级BALB／c小鼠宜用9、10周龄的进行超数排卵处理以供应3．5d胚胎为需要所用,而SPF级KM小鼠使用4～10周龄的都可以;另外,SPF级小鼠超数排卵处理仍宜在春、夏季进行,可以保证获得最佳的效果。     

Hr突变体转基因小鼠的构建和表型分析

无毛基因（Hr）定位于8p12,在染色体上跨越14 kb,包含19个外显子。无毛基因的自发突变能引起人和动物毛发脱落及相关毛发疾病的产生。为深入研究Hr基因的功能,本文利用Gateway技术构建Hr表达载体,在该基因的3 427位引入点突变(G→A),通过显微注射建立转基因小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6小鼠回交后互交数代建系。观察转基因小鼠毛发生长发育规律。结果表明,成功构建了pRP(Exp)-EF1A>mHairless mutant>IRES/EGFP真核表达载体,通过与野生型小鼠杂交获得阳性子代,进行同窝交配,第2代小鼠出生后14 d开始脱发,30 d左右脱落的毛发重新长出。取部分皮肤组织做石蜡切片,皮肤组织学观察发现,脱毛期无毛小鼠毛囊瓦解,真皮内形成大小不等的包囊,毛发重新生长时,真皮内见大量新生的毛囊。蛋白印迹实验表明,转基因小鼠脱发时HR蛋白表达量明显高于同龄阴性小鼠。本文成功建立稳定遗传的Hr突变的转基因小鼠品系,推测无毛基因突变引发转基因小鼠的脱发,为研究Hr基因的功能提供了良好的动物模型。     

昆明小鼠精子冷冻的研究(简报)

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受体,生长发育并产出幼仔。胚胎体外培养获得足够的晚桑葚胚和早期囊胚是胚胎干细胞建立与制备嵌合体小鼠的成功关键,暂时不用的胚胎应仍能耐受冷冻保存,待复苏移植,建立新的繁殖群。但小鼠精子冷冻研究在我国开展的十分有限,缺乏相关资料数据。本实验对不同周龄SPF级昆明(KM)小鼠进行精子冷冻及冻融精子IVF,并将IVF获得的2-细胞胚胎分别进行胚胎移植、体外培养、胚胎冷冻及复苏后移植,应用冻融小鼠精子进行胚胎工程实践。     

小鼠腹部心脏移植模型的制作与改进

目的建立并改进小鼠腹部异位心脏移植模型,为器官移植研究提供技术支持。方法SPF级近交系小鼠112只进行心脏移植手术,在传统方法的基础上进行改进,观察改进后手术效果并分析其优势。结果手术成功率为89.28％。总手术时间（103.9±16.5）min,受体手术时间（73.0±7.9）min。改进的方法简化了操作步骤,降低了手术难度。结论改进的小鼠腹部异位心脏移植技术是一种简便、有效、成功率高的模型制作方法。     

小鼠白内障基因突变方式、分布及基因定位

哺乳动物发育相关的功能基因的鉴定多来源于自发或诱发突变的小鼠。小鼠白内障作为较易鉴定的性状,目前业已明确的突变多达140余个。自发突变的小鼠主要来源于大规模饲养,诱发突变主要通过X射线与ENU处理获得,基因敲除与转基因小鼠亦可获得白内障性状。已知的白内障相关基因分布于小鼠20条染色体,其中以1号染色体最多,并常于小鼠胚胎时期起始表达。小鼠白内障表型多由单基因突变引起,突变的确定主要通过F2代家系全基因组扫描、精细定位、单倍型分型与测序验证等程序。本文从小鼠白内障基因突变来源、基因分布、基因定位与基因表达等角度较为全面的阐述了小鼠先天性白内障的研究进展。     

昆明小鼠无毛基因cDNA全长序列的分子克隆

无毛基因是与皮肤和被毛结构有重要关联的核受体基因,编码一个锌指结构转录因子,是甲状腺激素受体的转录辅阻遏物,并参与毛发生长周期的调控,在维持毛囊及毛囊间上皮的增殖、分化、调亡的精致平衡中扮演重要角色。参考小鼠、人的无毛基因序列,用RT-PCR方法首次对昆明小鼠无毛基因的cDNA序列进行了克隆,获得了昆明小鼠无毛基因的全长4 014 bp cDNA序列（GenBank 登录号：AY547391）,基因的CDS长度为3 546 bp。AY547391基因编码的蛋白质在GenBank中的序号为AAT45233,由1181个氨基酸组成。昆明小鼠与国外报导的小鼠、大鼠、猪、羊、猴、人等6种动物CDS 序列同源性分别为99.9%、94.4%、83.1%、78.1%、81.9%、82.1%,氨基酸同源性分别是99.9%、92.2%、81.7%、 70.8%、 79.9%、 80.1% 。这一结果反应了无毛基因在进化过程的高度保守性。通过Blast比较昆明小鼠无毛基因与GenBank 数据库中收集的Hr 基因的mRNA,发现了4个SNP和一个缺失突变,其中3个位点没有改变氨基酸残基的性质,两个位点有氨基酸改变并证实为多态性突变位点,研究结果为无毛基因的SNPs的数据库提供了新的信息。     

SPF级KM小鼠与BALB/c小鼠肠道总菌多样性的比较

目的分析SPF级封闭群KM小鼠及近交系BALB/c小鼠的肠道菌群总菌多样性,比较两个不同遗传背景肠道总菌的丰富度、Shannon-Wiener指数和均匀度。方法收集SPF级KM小鼠和BALB/c小鼠新鲜粪便,提取粪便总菌DNA,用基于细菌16S rDNA序列的变性梯度凝胶电泳(PCR-DGGE)分析粪便总菌多样性。结果 SPF级KM小鼠及BALB/c小鼠粪便总菌多样性差异无统计学意义(P0.05),品系内不同性别之间粪便总菌多样性差异亦无统计学意义(P0.05)。结论选择SPF级小鼠进行微生态学相关研究时,封闭群KM小鼠及近交系BALB/c小鼠均可作为选择对象,同时可忽略菌群的性别差异。     

BALB/c突变无毛小鼠特异性免疫功能的研究

目的　BALB c突变无毛小鼠的皮肤和被毛结构的突变是否影响机体的免疫功能 ,为此研究其免疫功能。方法　通过流式细胞仪和ELISA方法。对特异性免疫指标CD4+ 、CD3+ 、CD8+ 、CD19+ 、IgG进行检测。结果　各项指标雌雄之间差异不显著 ,无毛小鼠的各项指标均低于其他表型的指标。各组之间方差分析结果 :CD8+ 差异显著 ,其他指标差异不显著。IgG抗体的吸光度的结果 ,三种表型之间差异不显著。结论　该小鼠的皮肤和被毛突变对免疫功能有一定的影响。     

生物工程技术在实验小鼠生物净化上的应用

目的采用体外受精的技术,对L858R、TL清洁级小鼠,以及来源于野外的中华小家鼠,和感染肺炎克来伯氏菌的Balb/c-nu裸鼠进行生物净化。方法对于需要净化的小鼠的雄鼠,采集附睾的精子,放入HTF溶液中获能,然后加入经过超排的卵团,体外受精。20-22 h后,挑选形态正常的二细胞胚胎,在净化实验室,移植给假孕的SPF级ICR母鼠,待产仔。仔鼠断奶后,随机选择仔鼠及带奶母鼠送检。结果体外受精的胚胎,经过移植后,均顺利产仔;仔鼠及母鼠的微生物级别,均达到SPF级。结论对于微生物级别较低的实验小鼠,采用在洁净实验室内做体外受精、胚胎移植的方法,可以提高实验小鼠的微生物级别。     

Zygosity Determination in Hairless Mice by PCR Based on Hrhr Gene Analysis

We analyzed the Hr gene of a hairless mouse strain of unknown origin (HR strain, http://animal.nibio.go.jp/e_hr.html) to determine whether the strain shares a mutation with other hairless strains, such as HRS/J and Skh:HR-1, both of which have an Hr^hr allele. Using PCR with multiple pairs of primers designed to amplify multiple overlapping regions covering the entire Hr gene, we found an insertion mutation in intron 6 of mutant Hr genes in HR mice. The DNA sequence flanking the mutation indicated that the mutation in HR mice was the same as that of Hr^hr in the HRS/J strain. Based on the sequence, we developed a genotyping method using PCR to determine zygosities. Three primers were designed: S776 (GGTCTCGCTGGTCCTTGA), S607 (TCTGGAACCAGAGTGACAGACAGCTA), and R850 (TGGGCCACCATGGCCAGATTTAACACA). The S776 and R850 primers detected the Hr^hr allele (275-bp amplicon), and S607 and R850 identified the wild-type Hr allele (244-bp amplicon). Applying PCR using these three primers, we confirmed that it is possible to differentiate among homozygous Hr^hr (longer amplicons only), homozygous wild-type Hr(shorter amplicons only), and heterozygous (both amplicons) in HR and Hos:HR-1 mice. Our genomic analysis indicated that the HR, HRS/J, and Hos:HR-1 strains, and possibly Skh:HR-1 (an ancestor of Hos:HR-1) strain share the same Hr^hr gene mutation. Our genotyping method will facilitate further research using hairless mice, and especially immature mice, because pups can be genotyped before their phenotype (hair coat loss) appears at about 2 weeks of age.     

三种胚胎移植技术方法比较

以近交系FVB、DBA／2小鼠作为供体胚小鼠,封闭群CD-1小鼠作为受体小鼠,对实验小鼠胚胎移植的三种方法进行比较,即：对小鼠受精卵1细胞期胚(或2细胞期胚)经由受体小鼠输卵管口移植、经由受体小鼠输卵管壁移植及对小鼠进行子宫移植(8细胞期胚)的方法进行了实验研究,并对各方法适用于使用的动物对象、实验要求、产仔率、优势与不足等做了进一步探讨,为今后的实验动物提供参考依据。结果表明,输卵管壁移植优于输卵管伞口移植和子宫移植。     

Eliminating murine norovirus,Helicobacter hepaticus,and intestinal protozoa by embryo transfer for an entire mouse barrier facility

Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.     

BALB/c-nu裸小鼠繁殖中杂合仔鼠剔除时间对纯合裸仔鼠生长发育的影响

目的研究屏障设施下裸小鼠繁殖生产时,在不同时间段剔除杂合仔鼠对纯合裸仔鼠的成活率及生长发育情况的影响。方法选取25窝头胎生BALB/c-nu新生裸小鼠按剔除杂合仔鼠的时间不同分成五组,以24h剔除组为对照比较纯合裸仔鼠生长发育及死亡率、离乳率的差异性,统计分析其生长发育过程中的体重增长等。结果剔除组生长发育及死亡率与对照组之间均存在差异性,其中14d剔除组和21d剔除组差异极显著。结论剔除杂合仔鼠的时间对纯合裸仔鼠的成活率及生长发育情况的有很大影响。为了保证SPF级裸鼠生长发育要求,生产繁殖出合格的实验用裸小鼠,剔除全部杂合仔鼠的最佳时间应该在新生仔鼠出生24h内,超过3d则会极大地影响纯合裸小鼠的正常生长发育。     

Analysis of transfer of microinjected zygotes in production of transgenic mice

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients’ pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.     

Curly bare (cub), a new mouse mutation on chromosome 11 causing skin and hair abnormalities,and a modifier gene (mcub) on chromosome 5

In the outcrossing of a new recessive mouse mutation causing hair loss, a new wavy-coated phenotype appeared. The two distinct phenotypes were shown to be alternative manifestations of the same gene mutation and attributable to a single modifier locus. The new mutation, curly bare (cub), was mapped to distal Chr 11 and the modifier (mcub) was mapped to Chr 5. When homozygous for the recessive mcub allele, cub/cub mice appear hairless. A single copy of the dominant Mcub allele confers a full, curly coat in cub/cub mice. Reciprocal transfer of full-thickness skin grafts between mutant and control animals showed that the skin phenotype was tissue autonomous. The hairless cub/cub mcub/mcub mice show normal contact sensitivity responses to oxazolone. The similarity of the wavy coat phenotype to those of Tgfa and Egfr mutations and the map positions of cub and mcub suggest candidate genes that interact in the EGF receptor signal transduction pathway.     

Mouse cloned from embryonic stem (ES) cells synchronized in metaphase with nocodazole

Full-term development occurred when nuclei from mouse embryonic stem (ES) cells, synchronized in metaphase with nocodazole, were fused with enucleated oocytes or nuclei of reconstituted eggs and again fused with the enucleated blastomeres of fertilized two-cell embryos using inactivated Sendai virus. Two surviving male mice were derived from undifferentiated ES cell nuclei, one from single nuclear transfer and another from serial nuclear transfer. Both were noticeably small and died within 24 hr of birth for unknown reasons. These findings demonstrate that nuclear transfer of ES cells using the fusion method produces young, as does the piezoelectric-actuated nuclear transfer. J. Exp. Zool. 289:139-145, 2001.     

Helicobacter sp. MIT 01-6451 infection during fetal and neonatal life in laboratory mice

Helicobacter sp. MIT 01-6451 has been detected in SPF mice kept in Japan. To characterize strain MIT 01-6451, its infection route during fetal and neonatal life and effects on pregnancy were investigated using immunocompetent and immunodeficient mouse strains (BALB/c, C57BL/6, and SCID). MIT 01-6451 was detected in the uterus, vagina, and mammary glands of 50% of infected SCID mice, whereas these tissues were all negative in immunocompetent mice. No fetal infections with MIT 01-6451 were detected at 16–18 days after pregnancy in any mouse strain. In newborn mice, MIT 01-6451 was detected in intestinal tissue of C57BL/6 and SCID mice at 9–11 days after birth, but not in BALB/c mice. The IgA and IgG titers to MIT 01-6451 in sera of C57BL/6 female mice were significantly lower than those of BALB/c mice. Although no significant differences in the number of newborns per litter were observed between MIT 01-6451-infected and MIT 01-6451-free dams, the birth rate was lower in infected SCID mice than in control SCID mice. The present results indicated that MIT 01-6451 infects newborn mice after birth rather than by vertical transmission to the fetus via the placenta and that MIT 01-6451 infection shows opportunistically negative effects on the birth rate. In addition, the maternal immune response may affect infection of newborn mice with MIT 01-6451 through breast milk.