三个近交系C57BL/6J小鼠群体微卫星遗传变异分析(英文）

应用微卫星遗传标记对近交系C57BL/6J(B6)小鼠遗传稳定性进行分析。用FAM标记的引物PCR扩增了来自北京和上海三个实验动物生产单位提供的三个B6小鼠群体共15个微卫星位点并进行分型。结果显示,所有位点均处于纯合状态,其中7个位点为多态位点。研究表明各B6群体虽然为高度近交群体,但不同生产单位维持的B6群体之间存在遗传分化。     

从C57BL/6J小鼠胚胎中分离ES细胞系(英文)

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｓ）是来源于小鼠早期胚胎的细胞系,它可以在体外大量培养而不失去其发育的多潜能性。ＥＳ细胞不仅可以用来制作转基因动物,而且能够作为载体进行基因打靶等多种研究。目前,国际上常用的胚胎干细胞系都是来源于１２９小鼠的胚胎。因而,有必要探讨用其它品系小鼠建立ＥＳ系。１９９１年,Ｌｅｄｅｒｍａｎｎ等人首次报道从Ｃ５７ＢＬ／６Ｊ小鼠胚胎中建成了ＥＳ细胞系,但是没有对建立的细胞系进行特性分析。国内柴桂萱等人虽然做了特性分析,但是他们所建细胞系的细胞直径较大,生长速度较慢,不同于常见的ＥＳ细胞系。我们从Ｃ５７ＢＬ／６Ｊ品系小鼠胚胎中共分离了四个ＥＳ细胞系,分别命名为ＣＥ１、ＣＥ２、ＣＥ３、ＣＥ４（Ｆｉｇ．１ａ＆ｂ）。这四个细胞系核型正常率均达到７０％以上。我们检查了ＣＥ２细胞的分化能力,当将ＣＥ２细胞注入同基因型小鼠的皮下后,获得的畸胎瘤（Ｆｉｇ．３）组织切片检查的结果表明：该细胞能够分化成多种组织（Ｆｉｇ．２）。我们也研究了ＥＳ细胞的嵌合能力,用ＩＣＲ小鼠胚胎作为受体胚胎,采用囊胚显微注射法构建嵌合鼠。在幸存的幼鼠中我们获得了来源于ＣＥ２细胞的嵌合鼠（Ｔａｂｌｅ１,Ｆｉｇ．４）。综     

C57BL／6J小鼠ES细胞系的建立及其特性分析

本文报道从C57BL/6J个鼠的囊胚中,建立了三个ES细胞系MESPU17,MESPU18,MESPU19。这些细胞的细胞学特征和强AKP反应,表明这三个细胞系具有干细胞的特征。这三个细胞系均为XY型,正常二倍体核型分别占70％、88％和59％。体外分化可形成简单类胚,体内分化可形成瘤块。与国际上通用的CCE和来自129/ter的ES细胞MESPU13相比,这三个细胞系的ES细胞较大;在体外培养时,生长较慢;细胞也较粘,进行显微注射时,很容易粘在注射针壁上。MESPU17,MESPU18进行了嵌合体制作,以BALB/c和昆明鼠的囊胚为受体,采用囊胚注射法未获嵌合体,但使用昆阴鼠的8细胞胚注射法和共培养法得到嵌合体。     

Utility of a C57BL/6 ES line versus 129 ES lines for Targeted Mutations in Mice

Inbred ES lines, though useful for generating targeted mutations in mice, are used infrequently. To appreciate the relative efficiency of inbred ES lines, a C57BL/6 ES line was compared with 129 strain ES lines for effectiveness in chimera formation leading to the establishment of targeted mutations in mice. Data from a transgenic facility spanning 7 years were collected. C57BL/6 ES cells injected into Balb/c embryos results in lower coat color chimerism than do 129 ES cells injected into C57BL/6 embryos. Combined data indicate that five independent targeted C57BL/6 clones should be injected as compared to three independent 129 clones to generate enough chimeras to effectively test for germ-line transmission. Thus, although less efficient than 129 ES lines, the C57BL/6 ES line is a relatively competent line and useful for the routine generation of targeted mutations in mice on a defined genetic background.     

链脲佐菌素诱导C57BL/6J小鼠2型糖尿病模型研究

目的建立与2型糖尿病(非胰岛素依赖型糠尿病、NIDDM)病人临床特征和发病过程相似的NIDDM动物模型.方法用高脂肪饲料喂养C57BL/6J雄性断乳小鼠3周,腹腔注射链脲佐菌素(STZ),继续喂养4周,测定给药前和给药后1、3、4周非空腹血糖、实验结束时非空腹胰岛素水平,观察胰腺形态学变化.结果喂养3周后(给药前)高脂饲料-STZ组及高脂饲料-柠檬酸组血糖浓度(7.0±0.39)mmol/L、( 6.8±0.45)mmol/L高于普通饲料-STZ组及普通饲料-柠檬酸组(5.3±0.40)mmol/L、(5. 4±0.39)mmol/L,P＜0.05;实验结束时,高脂饲料-STZ组血糖浓度(13 .1±2.01)mmo/L高于高脂饲料-柠檬酸(6.9±0.46)mmol/L、普通饲料-柠檬酸组(6.0± 0.46)mmol/L和普通饲料-STZ组(7.1±0.62)mmol/L(P＜0.05),各组间血浆胰岛素浓度、体重及饮水量差异无显著性,P＞0.05;实验过程中高脂饲料STZ组和柠檬酸组小鼠每天进食热量(64.49±9.2)kJ/只,(70.7±9.6)kJ/只, 显著高于普通饲料STZ组和柠檬酸组(52.7±7.9)kJ/只,(57.3±11.7)kJ/只;各组小鼠胰腺和胰岛细胞形态正常.结论高脂肪饲料和STZ是用C57BL/6J断乳幼鼠建立NIDDM模型所必须的,100mg/kg体重STZ对普通饲料小鼠血糖无影响;用高脂饲料和STZ 处理的小鼠血糖升高、胰岛素浓度正常,与NIDM病人临床特征和发病过程相似;C57BL/6J小鼠易得,建模方法简便,费用低,是在NIDDM实验研究中能广泛使用的较理想的非遗传性NID DM动物模型.     

小鼠动物实验方法系列专题(三) 三硝基苯磺酸诱导的C57/BL6小鼠结肠炎模型的建立方法

2,4,6-三硝基苯磺酸(TNBS)诱导的小鼠结肠炎模型是研究人类炎性肠病(inflammatory bowl disease,IBD)的主要手段之一,但在实际应用中,常用的C57/BL品系小鼠却对TNBS有较高耐受性,不易建模。本文主要介绍一种可以有效诱导C57/BL6小鼠TNBS结肠炎的方法,并对疾病评价指标进行了具体的描述。对基因工程小鼠IBD模型的研究具有重要意义。     

Genetic differentiation within the inbred C57BL/6J mouse strain

The C57BL/6J (B6) inbred mouse strain is commonly used in biomedical researches. However, some unexpected inconsistency was reported compared with previous studies, and in most cases, it can be attributed to environmental, epigenetic or stochastic differences. The goal of this study was to investigate the genetic stability of the B6 strain maintained in different breeders. B6 mice purchased from five Chinese commercial breeders were examined, and mitochondrial D-loop sequence and 18 microsatellite loci were genotyped. There is no difference in the D-loop sequences, but variations exist in the nucleic microsatellite markers. Combining the data from MGI_4.01, a significant divergence is observed among those mice. The present study indicates that different B6 mice share the common maternal lineage and are still inbred in each breeder, but subline divergence occurs.     

具高效种系嵌合能力的C57BL／6J 小鼠ES细胞系的建立

采用小鼠原代成纤维细胞作为饲养层,在含1×103单位白血病抑制因子（LIF）的DMEM高糖培养基中,建成了11个C57BL／6J小鼠的ES细胞系,成系率为9．6％。所建的11个ES细胞系中有5个核型正常率大于70％。这些细胞具早期胚胎细胞的特征,呈碱性磷酸酶阳性,具表达0014基因的特性5进行体内分化实验时能发生广泛的分化。通过嵌合体制作实验证明其中3个系具嵌合能力,并从中筛选出具高效种系嵌合能力的MESPU35细胞系,MESPU35细胞的克隆能达到种系传递,经过基因操作的细胞克隆,也保留了高效的嵌合能力。因此,MESPU35细胞可作为制作突变小鼠的有效载体。     

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

C57BL/6小鼠性别差异对髓鞘少突胶质糖蛋白诱导的实验性自身脑脊髓炎疾病的影响

多发性硬化是人类常见的中枢神经系统自身免疫性炎症致脱髓鞘疾病．流行病学研究发现,女性患者多于男性,其平均发病时间早于男性．实验性自身免疫性脑脊髓炎(EAE)与多发性硬化症有相似的临床症状和病理特征,是被广泛应用于人类疾病研究的动物模型．本实验利用髓鞘少突胶质糖蛋白MOG_33-35免疫C57BL/6小鼠建立EAE模型,观察29天．通过疾病评分发现雌雄小鼠在发病率、起病时间上均无明显差别,但雄鼠的发病症状明显比雌鼠严重．在其病理切片HE染色中观察到雄性小鼠中枢浸润的炎性细胞多于雌性小鼠,并且在LFB染色中同样观察到雄鼠脱髓鞘区域明显增大．对其发病高峰期中枢浸润细胞的染色分析时,可以发现雄性小鼠中浸润的CD4 T细胞及其亚群T_H-1和T_H-17细胞均有明显增加．这些都表明MOG_33-35免疫C57BL/6小鼠建立的EAE模型存在着性别差异的影响,这一发现为今后建立多发性硬化症的动物模型中动物性别的选择提供了一定的参考依据．     

Lactation-induced WAP-SV40 Tag Transgene Expression in C57BL/6J Mice Leads to Mammary Carcinoma

Two transgenic lineages were generated by directing the expression of SV40 T antigen to the mammary gland of inbred C57BL/6J mice using the whey acidic protein (WAP) promoter. In one lineage, WAPTag 1, multiparous female mice developed mammary adenocarcinoma with an average latency period of 13 months. The histopathological phenotype was heterogeneous, tumours occurred in a stochastic fashion, normal tissue was located next to neoplastic tissue, the mammary tumours usually developed and were remarkably similar to that observed in human cases. In addition, male and virgin females developed a poorly differentiated SV40 T antigen-positive soft tissue sarcoma, also at 13 months of age. In the other lineage, WAPTag 3, some parous females developed mammary tumours, but most mice succumbed to osteosarcomas arising from the os petrosum at 5.5 to 6 months of age and on necropsy, renal adenocarcinomas were also found. Appearance of these unexpected tumour types demonstrates the non-specific e...     

Transglutaminase 3 expression in C57BL／6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.     

高脂饮食加低剂量链脲霉素建立小鼠2型糖尿病模型

目的高脂饮食加低剂量链脲霉素（Streptozotocin,STZ）建立小鼠2型糖尿病模型。方法5周的雄性C57BL/6J小鼠,随机分为正常饲料组、正常饲料加STZ组、高脂饲料组和高脂饲料加STZ组。相应饲料喂养5周后,按照100 mg/Kg的剂量腹腔注射STZ,然后继续喂养4周。在第5周和第9周末测定小鼠的体重、收缩压、血糖、血胰岛素、血甘油三脂和胆固醇水平。结果STZ注射前各组小鼠的体重、血压、血糖、血胰岛素、血脂和血甘油三脂无明显差异（P〉0.05）。STZ注射后4周时,高脂饲料加STZ组小鼠的体重、血糖、血胰岛素、血压和血脂水平明显升高（P〈0.05）;而其他三组的这些指标无明显改变或仅部分升高。结论高脂饮食加低剂量链脲霉素可建立小鼠2型糖尿病模型,该模型具有人2型糖尿病的主要表型特征和相似的发病过程。     

C57BL/6J小鼠在构建2型糖尿病模型中体重和非空腹血糖浓度的异常变化

由北京市一实验动物生产单位购入近交系C57BL/6J(B6)和封闭群ICR(3周龄)小鼠,分别以高脂饲料、高脂饲料-3%果糖饮水(实验组)和常规饲料(对照组)喂养6周,实验组腹腔注射链脲佐菌素(STZ,100mg/kg体重),然后以相应饲料继续喂养4周。每周测定小鼠体重,于注射STZ前和注射后每周测定非空腹血糖浓度。研究显示,无论是否补充果糖饮水,B6对照组体重显著高于实验组,而相应周龄的ICR小鼠,实验组体重显著高于对照组。两品系小鼠实验组间体重无差异。注射STZ后,B6实验组血糖浓度均没有达到糖尿病小鼠非空腹血糖浓度的成模标准(11mmol/L),而ICR实验组血糖浓度均达到并超过糖尿病小鼠非空腹血糖浓度的成模标准。研究表明,无论补充果糖与否,ICR小鼠均能成功建模,而B6小鼠建模均失败。因此,ICR小鼠仍是目前应用高脂饲料-STZ联合诱导2型糖尿病模型中经济、有效的候选动物,而B6小鼠在体重和血糖浓度上的异常表现很可能是其遗传背景变化的结果,这尚需进一步研究证实。     

Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media

C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background-however, relatively few such lines exist. Using a defined media supplement, knockout serum replacement (KSR) with knockout DMEM (KSR-KDMEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and after gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the generation of ES cells from inbred mouse strains.     

H5N1禽流感病毒对C57BL／6小鼠的致病性研究

本试验利用虎源H5N1禽流感病毒对6～8周龄雌性C57BL/6小鼠进行滴鼻感染,观测小鼠临床症状和组织病理变化,于感染后第3d和第5d每组分别处死3只小鼠,测定肺、脑、脾、肾、肝组织中的病毒含量;并测定该病毒对C57BL/6小鼠的MLD50。结果表明感染小鼠出现精神不振、体重下降、支气管炎和间质性肺炎为主的临床症状和病理变化;测得感染后小鼠肺脏中病毒拷贝数和病毒滴度最高,其次是脑、肾、脾、肝等组织;该病毒对C57BL/6小鼠的MLD50为10-6.5/0.05mL。此研究成功进行了H5N1禽流感病毒对C57BL/6小鼠的感染,可以作为感染模型进行H5N1禽流感病毒的发病机制、疫苗评价、药物筛选等研究。     

Proteins linked to extinction in contextual fear conditioning in the C57BL/6J mouse

Studying fear extinction is a major topic in neuroscience. No information on systematic studies on the linkage of contextual fear conditioning (cFC) with hippocampal protein levels is available and we were therefore interested in protein differences between animals with poor and good extinction. cFC was carried out in C57BL/6J mice, hippocampi were taken and proteins were run on two‐dimensional gel electrophoresis with subsequent quantification of protein spots. In‐gel digestion with trypsin and identification by ion trap MS/MS (high‐capacity ion trap) was used for the identification of significantly different hippocampal proteins between mice with good and poor performance of extinction. Signaling protein ras‐related protein rab‐7A and septin 8 levels were significantly higher in hippocampus of poor extinguishers, whereas ubiquitin carboxyterminal hydrolase isozyme L1 showed higher levels in animals with good extinction performance. A series of additional proteins showed significantly different levels between groups but the abovementioned were confirmed by immunoblotting. The abovementioned proteins have never been reported to be linked to extinction, memory, or learning and herein evidence for the involvement of several proteins in extinction mechanism as well as probably representing pharmaceutical targets is provided. Moreover, it is intriguing to demonstrate the differences between good and poor extinction performance at the protein level.