Beige-Nude小鼠免疫学特性研究概况

免疫缺陷动物是研究免疫相关疾病的良好动物模型。Beige-Nude小鼠是T淋巴细胞和NK细胞同时缺陷的小鼠,其T细胞和NK细胞活性很低,最初为肿瘤的研究而培育。从上世纪七十年代起,学者们对其免疫学特性进行了广泛而深入的研究,为其在其他领域的应用奠定了免疫学基础。     

乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠的制作与鉴定

目的利用Cre．LoxP重组酶系统构建乳腺上皮细胞特异性敲除Serib基因杂合子小鼠,并进行鉴定,为进一步在动物整体水平研究Scrib基因在乳腺癌中的作用提供研究平台。方法将Scrib条件敲除杂合子小鼠（Scrib＋/ft小鼠）进行繁殖并鉴定,然后将鉴定结果为阳性的子代Scrib＋/ft小鼠与乳腺上皮细胞特异性表达Cre重组酶的MMTV．Cre纯合子小鼠进行杂交,鉴定其子代小鼠的基因型。结果成功繁育Scrib条件敲除小鼠和MMTV．Cre小鼠,并通过鉴定得到Scrib＋/ft小鼠,与MMTV-Cre小鼠杂交并繁殖,获得基因型为Scrib＋/ft;MMTVCre＋/-小鼠5只。结论本研究利用Cre．LoxP重组酶系统成功构建了乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠,为进一步研究极性蛋白Scrib表达下调在乳腺癌发生中的作用提供了良好的动物模型。     

Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period

Viral, bacterial and parasitological infections in rats and mice used in biomedical research continue to occur despite improved housing and biosurveillance. The presence of disease in laboratory animals can lead to spurious results for research undertaken in universities, research institutes and the pharmaceutical industry. Here the authors report the results of serological, microbiological, parasitological and molecular tests done on mice and rats from Australasia submitted to a rodent health monitoring laboratory (Cerberus Sciences) from 2004 to 2009. In tested mice, norovirus was the most prevalent virus and ectromelia virus was the least prevalent virus. In tested rats, pneumonia virus of mice was the most prevalent virus and adenoviruses 1 and 2 were the least prevalent viruses. In mice, Helicobacter hepaticus was the most prevalent bacterium, and in rats, Proteus spp. were the most prevalent bacteria. The most common positive helminthological finding in mice and rats was the presence of all pinworms (including Aspicularis spp. and Syphacia spp.). The most common positive protozoan findings in mice and rats were Chilomastix spp. and Trichomonads.     

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.     

基于四环素调控系统的ApoE-rtTA/TRE-HCV-C双转基因小鼠的制备

目的为了丰富现有的四环素调控系统的小鼠资源库,同时也为更好地研究目的基因HCV-C的作用机制提供一个活体的动物模型,制备基于四环素调控系统原理的调控部分ApoE-rtTA的转基因小鼠,与反应部分TRE-HCV-C转基因小鼠,相互交配制作出双转基因小鼠。方法应用显微注射法将构建好的两种基因片段ApoE-rtTA及TRE-HCV-C分别注入超排的昆明母鼠的受精卵,再植入假母输卵管,出生动物及其后代经PCR初步筛选出阳性,将两者及阳性后代交配产生携带5只转基因鼠再经Western blot和RT-PCR在RNA和蛋白水平上进一步鉴定。结果产生了3只整合有ApoE-rtTA基因的首见鼠和125只阳性子代,以及产生了5只整合有TRE-HCV-C基因的首见鼠和16只阳性子代。PCR及Southern Blot证实上述阳性鼠确有转基因整合。将两者交配产生携带5只双转基因鼠。结论成功制备了携带有ApoE-rtTA及TRE-HCV-C转基因小鼠,建立了分别携带有这两种基因的鼠群,以及同时携带有两种基因的双转基因小鼠,为四环素调控系统提供了一个良好的通用型的调控动物模型,同时也可利用四环素调控系统来研究HCV中的C基因对小鼠的作用,也是HCV-C基因功能研究及与肝细胞癌的关系的机制研究的一个有用工具。     

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。     

NOD/SCID小鼠模型在实验血液学研究中的应用

NOD/SCID（非肥胖糖尿病/重症联合免疫缺陷）小鼠是在SCID（重症联合免疫缺陷）小鼠的基础上与非肥胖性糖尿病小鼠（NOD/Lt）品系回交的免疫缺陷鼠。NOD/SCID小鼠既有先天免疫缺陷,又有T和B淋巴细胞缺乏,各种肿瘤细胞可以植入,且较少发生排斥反应及移植物抗宿主病（GVHD）,所以NOD/SCID小鼠逐渐成为血液学实验研究的有用工具。本文从NOD/SCID小鼠的生物学特性、建立人类白血病模型、干细胞移植、药物研究及NOD/SCID小鼠应用中存在的不足和改良等方面综合述评。     

提高制备转基因小鼠效率的研究

目的提高制备转基因动物的效率.方法着重对注射DNA的制备,高质量受精卵的获得,显微注射及胚胎移植各步骤中的主要影响因素进行了优化.结果移植后出生的小鼠经PCR及Sonthern-Blot检测,从最初的146只仔鼠中检测到2只阳性鼠,提高到由34只仔鼠中检测到10只阳性鼠,阳性鼠概率由1.4%基本稳定增加到30%.结论通过技术改良后,转基因效率得到明显提高,为从事转基因动物方面的研究建立了稳定的技术平台.     

基因修饰小鼠饲养管理及使用过程中问题探讨

随着基因修饰小鼠的广泛使用,在饲养管理中必然会遇到一些新问题。文章以北京大学医学部实验动物科学部清洁级动物实验设施中饲养的基因修饰小鼠为对象,着重探讨其在饲养管理和使用中出现的问题,并加以分析,为建立基因修饰小鼠的管理规范奠定基础。     

APP/PS1双转基因小鼠的繁育、鉴定及评价

目的：评价APP/PS1双转基因小鼠基因表达及认知行为能力的变化,为AD的相关研究提供有效的动物模型。方法：采用雄、雌鼠1：1合笼配对的方式,令APP/PS1双转基因小鼠自然交配进行繁育。PCR鉴定APP/PS1双转基因鼠仔鼠的基因型后,选择APP/PS1阳性小鼠作为模型（AD）组,同批APP/PS1阴性为对照（CT）组,每组8只小鼠。以Morris水迷宫实验检测仔鼠的空间学习记忆能力,以HE染色、刚果红染色观察仔鼠脑片组织病理学改变。结果：①APP/PS1双转基因鼠仔鼠基因经PCR扩增,出现约360 bp的目的基因条带,表明成功繁育出转入APP/PS1基因的仔鼠;②Morris水迷宫实验结果显示,与7月龄阴性小鼠（CT组）比较,同月龄的双转基因AD组小鼠的空间学习记忆能力明显降低（P<0.05）;③HE染色结果显示,AD组小鼠海马结构及细胞形态出现明显异常;刚果红染色结果显示,AD组小鼠脑片组织出现β淀粉样蛋白斑块沉积。结论：APP/PS1双转基因小鼠较好地模拟了AD的病理变化及行为学特征,可作为研究AD发病机制及开发AD防治药物的实验工具。     

Mice in Bion-M 1 Space Mission: Training and Selection

After a 16-year hiatus, Russia has resumed its program of biomedical research in space, with the successful 30-day flight of the Bion-M 1 biosatellite (April 19–May 19, 2013). The principal species for biomedical research in this project was the mouse. This paper presents an overview of the scientific goals, the experimental design and the mouse training/selection program. The aim of mice experiments in the Bion-M 1 project was to elucidate cellular and molecular mechanisms, underlying the adaptation of key physiological systems to long-term exposure in microgravity. The studies with mice combined in vivo measurements, both in flight and post-flight (including continuous blood pressure measurement), with extensive in vitro studies carried out shortly after return of the mice and in the end of recovery study. Male C57/BL6 mice group housed in space habitats were flown aboard the Bion-M 1 biosatellite, or remained on ground in the control experiment that replicated environmental and housing conditions in the spacecraft. Vivarium control groups were used to account for housing effects and possible seasonal differences. Mice training included the co-adaptation in housing groups and mice adaptation to paste food diet. The measures taken to co-adapt aggressive male mice in housing groups and the peculiarities of “space” paste food are described. The training program for mice designated for in vivo studies was broader and included behavioral/functional test battery and continuous behavioral measurements in the home-cage. The results of the preliminary tests were used for the selection of homogenous groups. After the flight, mice were in good condition for biomedical studies and displayed signs of pronounced disadaptation to Earth''s gravity. The outcomes of the training program for the mice welfare are discussed. We conclude that our training program was effective and that male mice can be successfully employed in space biomedical research.     

Mice Drawer System: phase c/d development and perspective

MDS (Mice Drawer System) is the Facility that will support the research on board the International Space Station (ISS). Funded by ASI with Laben as industrial Prime Contractor, MDS will also permit its utilisation for other research programs with mice. The most attractive feature of MDS is that six mice can he kept separate inside dedicated and isolated cages. This permits to perform six experiments in parallel, one for each mouse. In the first utilisation flight of MDS facility the selected experiment is devoted to study human bone formation and specific countermeasures to prevent osteoporosis using mice under microgravity conditions as a model.     

胚胎移植技术对特种突变小鼠的净化研究

目的通过净化特种突变小鼠来建立SPF级种子库。方法利用胚胎移植技术对四种突变无毛小鼠和一种白内障小鼠的桑葚胚进行子宫内移植。结果四种突变无毛小鼠和白内障小鼠均产出仔鼠,获得移植成功,其中白内障小鼠的产仔率最高为25.5%。结论建立了SPF级特种突变小鼠种群,为进一步研究与开发利用提供了可靠保证。     

基因剔除小鼠研究的新进展

基因剔除小鼠是活体内研究基因及蛋白质功能的理想动物模型。传统的基因剔除技术存在表型分析复杂、周期长、操作繁杂等缺点,如何更科学、更方便地建立基因剔除小鼠模型成为目前使该技术更好地应用于医学研究的关键。本文从胚胎干细胞（ES细胞）的遗传背景、组织特异性基因表达基因剔除小鼠及基因剔除技术上的革新几方面介绍了目前基因剔除小鼠研究的一些最新进展。     

Rodent and germplasm trafficking: risks of microbial contamination in a high-tech biomedical world

High-tech biomedical advances have led to increases both in the number of mice used for research and in exchanges of mice and/or their tissues between institutions. The latter are associated with the risk of dissemination of infectious agents. Because of the lack of international standardization of health surveillance programs, health certificates for imported rodents may be informative but may not address the needs of the importing facility. Preservation of mouse germplasm is achieved by cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells are used for the production of genetically engineered mice. After embryo transfer, recipients and rederived pups that test negative in microbiological screening for relevant microorganisms are released into full barrier holding areas. However, current research shows that embryos may also transmit microorganisms, especially viruses, to the recipient mice. In this article, we discuss regulations and practical issues in the shipping of live mice and mouse tissues, including spermatozoa, embryos, ovaries, and embryonic stem cells, and review work on microbial contamination of these biological materials. In addition, we present ways to reduce the risk of transmission of pathogens to mice under routine conditions.     

Inbred mouse strains and genetic stability: a review

Inbred mice were essential animal models for scientific research during the 20th century and will contribute decisive results in the current and next centuries. Far from becoming an obsolete research tool, the generation of new inbred strains is continuing and such strains are being used in many research fields. However, their genetic properties have been overlooked for decades, although recent research has revealed new insights into their genetic fragility and relative instability. Contrary to what we usually assume, inbred mice are far from being completely isogenic and both single-gene major mutations and polygenic mutational variability are continuously uploading into inbred populations as new sources of genetic polymorphisms. Note that several inbred strains from new major mutations are released every year, whereas small mutations can accumulate up to accounting for a significant percentage of the phenotypic variance (e.g. 4.5% in a recent study on C57BL/6J mice). Moreover, this genetic heterogeneity can be maintained for several generations by heterozygote selection and, if fixed instead of dropping off, genetic drift must be anticipated. The contribution of accidental genetic contamination in inbred strains must also be considered, although its incidence in current breeding stocks should be minimal, or even negligible. This review revisits several relevant topics for current inbred strains, discussing the latest cutting-edge results within the context of the genetic homogeneity and stability of laboratory mice. Inbred mice can no longer be considered as completely isogenic, but provide a remarkably homogeneous animal model with an inevitable moderate-to-low degree of genetic variability. Despite a certain degree of genetic heterogeneity becoming inescapable, inbred mice still provide very useful animal models with evident advantages when compared with outbred, that is, highly variable, populations.     

人G-CSF转基因鼠的稳定遗传与表达

利用人粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因组基因作为目的片段,将其受控于２．６ｋｂ的小鼠乳清酸蛋白（ＷＡＰ）基因的调控区下,通过显微注射法获得了两只整合有人Ｇ－ＣＳＦ转基因小鼠,通过繁殖建立了稳定的转基因系．一些表型参数测定表明转基因鼠与正常鼠无明显差别．通过ＲＴ－ＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,在乳腺表达出人Ｇ－ＣＳＦ,为乳腺表达外源蛋白质及今后大动物研究奠定了基础．     

Urinary excretion of uric acid,allantoin, and 8-OH-Deoxyguanosine in uricase-knockout mice

ABSTRACT

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography–mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.     

S100a4-Cre×Ddr2flox/flox条件敲除小鼠的繁育及基因型鉴定

目的:利用Cre-LoxP重组酶系统构建成纤维细胞中Ddr2特异性敲除的纯合子小鼠并鉴定,为进一步研究Ddr2在肺纤维化中的作用提供基础。方法:将购买的Ddr2 loxp小鼠和成纤维细胞特异表达的S100a4-Cre小鼠分别繁殖与鉴定,然后将两种小鼠杂交与鉴定,最终得到基因型为Cre~× Ddr2~(flox/flox)的纯合子小鼠就是在成纤维细胞中Ddr2条件性敲除小鼠。结果:成功繁殖并用PCR技术准确鉴定了基因型为Cre~× Ddr2~(flox/flox)的纯合子小鼠,Western blot结果表明纯合子小鼠的肺成纤维细胞中Ddr2已缺失。结论:本研究利用Cre-LoxP系统成功构建了在成纤维细胞中Ddr2特异性敲除的纯合子小鼠,为进一步研究Ddr2在肺纤维化发展中的机制提供了研究平台。     

基因改造家兔在动脉粥样硬化研究中的应用

目前应用转基因和基因敲除小鼠的研究工作几乎成为各种国际高水平学术刊物的主流,而基因改造的家兔由于其独有的特点,在心血管研究中占有重要地位。本文从基因改造小鼠的最新研究进展着手,分析了基因修饰小鼠在动脉粥样硬化研究中的局限性,进而阐明基因改造家兔用于动脉粥样研究的优越性及研究近况。文章分析表明基因改造家兔技术及其应用必将在生物医学研究中发挥重要作用。