应用显微注射法建立转基因小鼠

应用显微注射法建立转基因小鼠谢卫兵陈汉源曾位森罗琛（第一军医大学组胚教研室广州５１０５１５）转基因动物是研究基因表达调控及表达产物生物学效应的最佳体系之一，同时作为一种新型的生物反应器，在医学、农、林、畜牧业等领域有着广泛的应用前景。目前采用基因转移...     

双重引物PCR鉴定APPSWE转基因小鼠

本实验旨在探讨一种简单易行,能有效识别APPSWE转基因小鼠基因检测中假阴性结果的方法。在PCR体系中设计两对引物,一对为APP基因特异性引物,另一对为根据其内源性管家基因β-actin设置的参照引物。利用同一个PCR反应体系,对两对引物进行扩增。结果显示,双重引物PCR扩增的特异性片段与单引物扩增片段完全吻合,利用该法检出的转基因阳性率高于单引物PCR检出的转基因阳性率。本方法简单易行,能够有效识别以往利用单一PCR检测出现的假阴性结果,并且具有很好的特异性和灵敏性,值得在转基因小鼠鉴定实验中推广。     

人突变TDP-43转基因小鼠ALS模型的建立

目的 构建带有脊髓运动神经元特异性启动子HB9的人类TDP-43野生型和突变型的转基因小鼠模型,研究TDP-43突变导致肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)的机制.方法 构建pHB9 - hTDP -43Q331K,M337v - IRES- EGFP转基因的载体,通过受精卵原核注射的方法制备转基因小鼠.出生的后代利用PCR方法鉴定.免疫组织化学法观察转基因小鼠的脊髓运动神经元中TDP-43的定位及包涵体的形成.采用步态评估和悬挂实验观察转基因小鼠行为学的改变.结果 获得人TDP-43突变转基因小鼠,其运动神经元胞质中TDP-43染色阳性,未见TDP-43包涵体.小鼠行为学检测显示,转基因小鼠的步距与同龄正常小鼠相比明显下降(Q331K型小鼠同Ntg相比前后肢步距t值分别为6.05和5.15,M337V型小鼠同Ntg相比前后肢步距t值分别为10.71和9.91;P＜0.01);步态宽度升高(Q331K型小鼠同Ntg相比前后肢步态宽度t值分别为-11.35和-2.73;M337V型小鼠同Ntg相比后肢步态宽度t=2.49;P＜0.05);平衡抓握力明显下降(Q331K型小鼠同Ntg相比落地时间t=47.43;M337V型小鼠同Ntg相比落地时间t=26.35,P＜0.01).结论 利用HB9启动子可以获得在脊髓运动神经元特异性表达人TDP-43突变的转基因小鼠,TDP-43突变转基因小鼠运动神经元发生退行性改变.转基因小鼠前后肢步距、步态宽度及平衡抓握能力发生改变,结果显示脊髓运动神经元中过表达人TDP-43突变对小鼠的运动能力产生影响.     

Alzheimer病转基因小鼠模型的研究进展

Ａｌｚｈｅｉｍｅｒ病 (Ａｌｚｈｅｉｍｅｒ’ｓｄｉｓｅａｓｅ,ＡＤ)是一种进行性的精神衰退的疾病 ,又称早老性痴呆。在老年人中ＡＤ的发病率很高 ,而且 ,随着年龄的增长呈指数性增加。世界上 6 5岁以上人群中ＡＤ的发病率在 5 % - 10 % ,在 85岁以上的老年人中发病率约为 2 5 % ,其中 ,10 %的患者具有明显的ＡＤ家族史 ,在这些家族性ＡＤ患者中 ,5 % - 10 %的病例具有常染色体显性遗传的特点[1] 。ＡＤ临床表现为慢性进行性的病变过程 ,发病早期出现短期记忆力丧失 ,随后 ,发展为进行性痴呆 ,主要表现为记忆力丧失 ,定向障碍 ,判断和推理…     

乙肝成体转基因小鼠模型的建立及应用

乙型肝炎病毒(hepatitis B virus,HBV)属于嗜肝DNA病毒科,其感染可导致人类急慢性肝炎,尽管预防性乙肝疫苗的广泛接种,极大的降低了HBV感染的发病率,但由慢性乙型肝炎及其导致的肝硬化和肝细胞癌等仍严重危害人类的健康[1].     

人Man2c1转基因小鼠模型的建立

目的 为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法 构建pIRKS2-EGFP-hMan2c1重组表达载体，经体外转染实验鉴定转染的基因能在COS-7细胞表达后，注射人ICR小鼠受精卵，以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Westernblot分析hMan2c1在转基因小鼠的表达。结果 在116只原代小鼠中，有7只hMan2c1基因组PCR阳性。在所检测的20只F1代小鼠中，有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中，有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达，确定基因组PCR阳性的7个系中有4个系阳性。结论 建立了4个稳定表达hMan2c1的转基因小鼠系，为深入研究MAN2C1的生物学意义打下了基础。     

小鼠子宫内电转基因方法的建立

目的:建立小鼠子宫内电转基因技术,比较分析转染绿色荧光蛋白(GFP)后对胚胎发育及相关蛋白表达的影响.方法:将怀孕15d的小鼠,水合氯醛麻醉后,取出两侧子宫,用毛细管注射针将2μg/μl的pCAGGS-GFP质粒0.5～1 μl准确注射到胎鼠侧脑室,在电压40 V、每次脉冲60 ms,间隔940 ms,电脉冲6次的条件下进行定时定位活体电转基因,电转后24 h取材,甲醛固定冷冻冠状切片,DAPI染细胞核观察组织形态结构变化,荧光免疫组织化学检测α-SMA的表达差异.结果:妊娠15d孕鼠转染24 h后小鼠成活率80％(8/10),胚胎成活率为54.2％(13/24),存活胚胎GFP阳性表达率为61.5％(8/13),GFP阳性表达胚胎脑组织切片,基因转染区域和正常组织区组织形态结构和α-SMA表达不存在差别.结论:成功建立了小鼠子宫内电转基因的方法.     

转基因小鼠研究进展

转基因动物是指用实验的方法将外源基因导入早期胚胎细胞,使之整合于细胞基因组中而建立的动物品系.最早建立成功的转基因动物是转基因小鼠(transgenic mice).由于转基因小鼠的建立比其它大型转基因哺乳动物的建立要省时、省力,因此转基因小鼠作为生命科学研究的一个新体系已经得到越来越广泛的应用.     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

Mito-cpYFP cDNA转基因小鼠的建立及其自由基分布规律的初步探讨

目的:利用可实时动态测量超氧阴离子自由基的环状排列黄色荧光蛋白(circularly permuted yellow fluorescent protein,cpYFP)构建可实时动态监测活体动物体内自由基的转基因动物模型。方法:应用转基因技术将载有可定位于线粒体内的cpYFP(mitochondria-targeted cpYFP,mito-cpYFP)的载体pRP(Exp)-CAGGmitocpYFP注射到C57BL/6小鼠的受精卵中,采用PCR及RT-PCR的方法鉴定阳性小鼠,通过荧光观察自由基的分布并辅助鉴定阳性小鼠。结果:PCR、RT-PCR及荧光观察结果表明mito-cpYFP cDNA转基因小鼠构建成功。通过与野生型C57BL/6小鼠交配,得到F1、F2和F3代的新生幼鼠共计494只,其中,阳性幼鼠为255只,阳性率为51.6%。转基因阳性鼠体内出现了绿色荧光,其解剖伤口、肠道、脑部及口腔黏膜等荧光明显,而肾脏、肝脏及胸腺等荧光则较弱,耻骨联合部和膀胱出现绿色荧光,但约0.5 h后消失。结论:我们成功制备了可稳定表达并遗传mito-cpYFP的转基因小鼠品系。在活体动物体内,自由基在黏膜层含量较多。此模型可研究自由基在生物体内的含量及分布规律,为后续生物自由基作为信号分子在机体内传导途径的研究提供有效的工具。     

心肌组织特异性高表达核仁素转基因小鼠的构建与鉴定

 目的：构建并鉴定心肌组织特异性高表达核仁素(Ncl)的转基因小鼠,为从整体水平上研究核仁素对心肌功能的影响及其发挥心肌保护作用的机制提供动物模型。方法：采用基因重组法将小鼠核仁素基因的全长cDNA插入含心肌细胞特异性启动子的Alpha-MyHC clone 26载体,构建Alpha-MyHC clone 26-Ncl重组载体,通过双酶切及基因测序鉴定其正确性;采用PCR鉴定阳性小鼠并用Western blotting法观察转基因小鼠中核仁素的表达情况,采用组织切片及HE染色法观察转基因小鼠心肌组织形态;测量心脏重量指数和左室压最大上升速率,观察核仁素在心肌组织高表达后对小鼠心脏形态功能是否有影响。结果：PCR法观察显示获得4只阳性小鼠(51、52、56和86,其中52和86系繁殖良好）,Western blotting结果表明转基因小鼠的核仁素表达量明显高于野生鼠,HE染色等结果表明核仁素对心肌形态学、心脏/体重比值及心功能无明显影响。结论：成功制备了心肌组织特异性高表达核仁素的转基因小鼠,与野生型小鼠相比,转基因小鼠心肌形态及心功能无明显差异。     

Interleukin-4 transgenic mice of resistant background are susceptible to Leishmania major infection

The outcome of cutaneous leishmaniasis is dependent on the balance of Th1 and Th2 cells. In the murine model, Th1 cells are host-protective whereas the Th2 cells are disease-promoting. However, the in vivo role of interleukin-4 (IL-4), a signature product of Th2 cells, is uncertain. We compared the course of Leishmania major infection in the genetically resistant 129/Sv mice and the mutant 129/Sv mice transgenic for the murine IL-4 gene under the control of the immunoglobulin heavy chain enhancer and promoter. We report here that in contrast to their wild-type parents, the IL-4 transgenic mice are susceptible to L. major infection. This is associated with the development of inexorably progressive lesions and parasite loads. Spleen cells from infected transgenic mice produced significantly higher levels of IL-4 but lower amounts of interferon-γ when stimulated in vitro with leishmanial antigens compared to those from infected normal 129/Sv mice. Furthermore, sera from the infected transgenic mice contained higher levels of IL-4 and IgE than the sera of infected normal 129/Sv mice. These results, therefore, establish in a new animal model that IL-4 promotes disease development in murine cutaneous leishmaniasis.     

阿尔茨海默病转基因小鼠海马结构NIX的变化

目的观察阿尔茨海默病转基因小鼠海马结构NIX的变化。方法以Morris水迷宫检测野生型和突变型转基因小鼠学习记忆能力,免疫组织化学和共聚焦激光扫描显微技术观察转基因小鼠海马结构促凋亡蛋白NIX的变化结果野生型和突变型小鼠逃避潜伏期中位数分别为29.00 s和38.00 s,差异无统计学意义,P>0.05;野生型和突变型小鼠搜索策略相比,突变型较野生型使用的搜索策略减少,差异有统计学意义,P<0.05;野生型和突变型小鼠NIX免疫反应阳性物灰度值中位数分别为103.83和128.85,差异有统计学意义,P<0.05;野生型和突变型小鼠海马结构NIX平均荧光强度分别为92.18±7.81和103.07±14.94,差异有统计学意义,P<0.05;野生型和突变型小鼠海马结构NIX与线粒体共定位的数目分别为240.94±169.48和544.18±336.44,差异有统计学意义,P<0.05。结论阿尔茨海默病转基因小鼠出现学习记忆障碍,海马结构促凋亡蛋白NIX的量增多,且NIX与线粒体共定位的量增多,提示NIX在阿尔茨海默病病理改变过程中可能起到一定的作用。     

Cataracts in transgenic mice caused by a human papillomavirus type 18 E7 oncogene driven by KRT1-14

Human papillomavirus type 18 (HPV18) is a common cause of cervical cancer. To create a mouse model for this common neoplastic disease, we used a human keratin 14 promoter to drive the HPV18 E7 oncogene to create transgenic mice. No mice up to a year of age developed cervical cancer. However, all transgenic mice and none of the controls developed progressive bilateral cortical cataracts. By 6 months of age, the cortex liquefied leaving the lens nucleus. Proliferation of lens epithelium formed multifocal nodules and free floating lens epithelial cells within the liquefied cortex. These cells were hyperplastic not neoplastic. Other HPV transgenic stocks develop cataracts suggesting this virus may have a broad cellular tropism.     

A strategy for the ubiquitous overexpression of human catalase and CuZn superoxide dismutase genes in transgenic mice

In the present study, we generated transgenic mice that overexpress catalase or CuZn superoxide dismutase (CuZnSOD) in all tissues using large genomic DNA fragments. An 80 kb human genomic DNA, containing the 33 kb human CAT gene as well as the 41 kb of 5' and the 6 kb of 3' flanking regions, was obtained by screening a human P1 library and was used to produce transgenic mice Tg(CAT). Transgenic mice Tg(SOD1) were produced by a similar strategy using a 64 kb human genomic DNA containing the 10 kb human SOD1 gene and the 27 kb of both 5' and 3' flanking regions. Catalase mRNA levels were 2-6- fold higher and catalase activity levels were 2-4- fold higher in the various tissues of the hemizygous Tg(CAT) mice compared with wild type mice. The mRNA levels for CuZnSOD were 2-12- fold higher and the CuZnSOD activity levels were 2-5- fold higher in the hemizygous Tg(SOD1) mice compared with wild type mice. In summary, our study demonstrates that a strategy of using large genomic DNA containing either the entire human CAT or SOD1 gene with large flanking regions gives ubiquitous increased expression of CuZnSOD and catalase. In addition, the expression of catalase closely reflects the tissue specific pattern found in the endogenous gene. These transgenic mice will be useful in studying the role of oxidative stress/damage in aging and age-related pathologies.     

The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci

An approach to the preparation of antigen-specific human monoclonal antibodies focuses on mice transgenic for human immunoglobulin gene miniloci; the V gene segments in these miniloci undergo productive rearrangement to yield mouse B cells expressing human immunoglobulin (Ig) chains. The general usefulness of this strategy hinges on whether it is feasible to obtain specific, high-affinity antibodies following immunization of such animals with a variety of antigens. To test this, we have investigated the antigen-specific responses in mice which carry human IgH miniloci (constaining just one or two V_H segments) instead of a functional mouse IgH locus. Although serum responses were relatively weak, monoclonal antibodies were readily obtained to all immunogens tested (a hapten, foreign proteins and human lymphoma cells). The affinities of two of the hapten-specific (anti-2-phenyl-oxazol-5-one) antibodies were 60 and 160 nM, values intermediate between what is typically obtained in the primary and secondary response of normal mice. Sequence analysis of the rearranged V genes revealed that junctional events made a major contribution to diversity with a considerable amount of apparently non-templated sequence at the V-D and D-J borders. Somatic hypermutation was also evident within the expressed V gene segments of many of the antigen-specific hybridomas. These findings augur well for the general usefulness of the transgenic approach for the isolation of high-affinity human antibodies to a wide range of antigens and suggests that the miniloci need not be particularly large.     

Genotype-related changes of ganglioside composition in brain regions of transgenic mouse models of Alzheimer's disease

In this study, brain gangliosides of different transgenic mouse models of Alzheimer's disease (AD) were analyzed and compared with age-matched wild-type mice. Gangliosides were analyzed in cerebral cortex, a region with extensive Aβ plaques, and cerebellum, a non-vulnerable region with no Aβ containing plaques. There was a marked increase in simple gangliosides GM2 and GM3 only within the cortex of all mice expressing APP^SL. Additionally, loss of complex “a” gangliosides (GT1a, GD1a and GM1) was recorded in APP/PS1Ki model, whereas in APP^SL and APP/PS1 mice, the complex “b” gangliosides (GQ1b, GT1b and GD1b) moderately decreased. Surprisingly, expression of either mutant PS1^M146L or PS1 mutant FAD (Ki model) alone tended to lower the levels of both GM2 and GM3 within the cortex. Conversely, only slight changes of the ganglioside pattern were found in the cerebellum. Because ganglioside alterations occurring in APP transgenic mice were similar to those observed in human AD brain, these transgenic models would represent valuable tools to further investigate the role of altered ganglioside metabolism in the pathogenesis of AD.