人β_2m转基因小鼠的制备及鉴定(英文)

制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

人β_2m转基因小鼠的制备及鉴定(英文)

 制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

TIMP-1转基因小鼠纯合子的建立及建系

采用遗传学育种方法 ,使外源基因整合位点随机的基质金属蛋白酶抑制剂 1(TIMP 1)转基因小鼠成为单一整合位点的纯合子转基因小鼠而建立TIMP 1转基因小鼠品系 .通过受精卵原核显微注射方法 ,获得带有人TIMP 1基因的Founder小鼠 .将转基因小鼠与正常小鼠交配 ,得到子代小鼠 .通过PCR及Southern印迹等方法 ,检测TIMP 1DNA在转基因小鼠体内的整合情况 ,阳性率达5 0 %后 ,进行近亲交配 .提取小鼠组织总RNA ,Northern印迹分析阳性小鼠各组织外源性TIMP 1mRNA表达情况 ,以正常NIH小鼠做对照 .获得了 6代小鼠共 4 2 4只 ,其中PCR阳性鼠 2 72只 ,Southern阳性鼠 2 2 6只 ,纯合子转基因小鼠 12 8只 ;F4代后阳性率达到 95 %以上 .转基因小鼠TIMP 1基因表达情况在肾脏的丰度明显高于肝脏和脾脏 (P <0 0 1) ,而肝和脾之间并没有显著差异 (P>0 0 5 ) .外源基因在转基因小鼠体内可以稳定遗传 ,并得到了整合有TIMP 1基因的纯合子转基因小鼠 ,且在阳性的转基因小鼠体内在肾脏中特异性表达 ,为以后开展TIMP 1的肾脏病理生理研究提供了有用的手段     

三种丙型肝炎病毒转基因小鼠系的同时制备

为研究丙型肝炎病毒的致病致瘤机理及结构基因与非结构基因3区(NS3)的功能及其在HCV感染致病中的作用,建立一个HCV分子治疗的动物模型,构建了含金属硫蛋白启动子和HCV结构基因或NS3基因的质粒,将两者等量混合后用显微注射法接种于昆明白小鼠受精卵内制备转基因小鼠.通过PCR筛选获得三种整合HCV结构基因或/和NS3基因的首建鼠.结果表明:a.注射后卵存活率与仔鼠出生率分别为81%、30%;b.检测60只G0代小鼠,结构基因整合鼠6只(10%),NS3基因整合鼠4只(6.7%),双基因整合鼠9只(15%),总整合率为31.7%;c.RT-PCR法检测阳性鼠肝中有靶基因mRNA的转录;d.4只首建鼠与正常鼠回交获得38只G1小鼠,其中20只为整合鼠,整合率为52.6%;e.转基因鼠表型迄今无明显异常.表明一次显微注射同时获得了三种整合HCV结构基因或/和NS3基因的转基因小鼠.     

携带HLA-B2704基因转基因小鼠技术的建立

应用显微注射法制备携带HLA B2 70 4基因的转基因小鼠 .对 2 86只昆明小鼠激素注射进行超排卵 ,采集受精卵 ,将含HLA B2 70 4基因的基因组DNA片段 (简称HLA B2 70 4DNA)显微注射到受精卵原核内 ,把注射存活的两细胞期受精卵移入假孕鼠的输卵管内使其发育产生后代 .用PCR方法进行F0代仔鼠及F1代仔鼠的转基因整合的检测 .利用RT PCR检测阳性鼠中的HLA B2 70 4转基因的表达 .采集了 84 11个卵 ,可注射卵 6 6 0 9个 ,其中注射存活的两细胞期受精卵 4 2 77个 ,卵的注射存活率为 6 4 7%.将卵移入 15 3只假孕鼠 ,其中 2 6只怀孕产仔 ,存活 10 1只 .在 10只F0代仔鼠基因组中有HLA B2 70 4基因整合 ,整合率为 9 9%.转基因阳性鼠F0代之间以及与正常鼠之间进行交配 ,产生的F1代仔鼠 78只 ,其中 15只为阳性 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA B2 70 4转基因mRNA的表达 .在HLA B2 70 4转基因阳性小鼠中 ,6只小鼠皮肤出现脱毛 ,1只小鼠的足部及足趾明显红肿 ,2只在脱毛同时明显畏光 ,1只出现腹泻 .结果表明 ,成功地建立了HLA B2 70 4的转基因小鼠技术 ,该小鼠类似强直性脊柱炎的小鼠模型 .     

携带EB病毒潜伏膜蛋白基因转基因小鼠的制备

EB病毒潜伏膜蛋白(LMP)具有致癌活性,构建含金属硫蛋白基因-1(MT-1)调控区和LMP基因的pBR322-MT-LMP质粒,用显微注射法将MT-LMP转基因接种于小鼠受精卵内制备转基因小鼠的动物模型.结果:a.注射后卵的存活率和仔鼠出生率分别为73%和13%;b.转基因鼠尾部组织DNA分析,PCR法证明二只小鼠有LMP基因扩增带,Southern杂交亦显示PCR阳性鼠有特异的LMP杂交信号,这说明LMP基因已整合于鼠基因组,整合率为8%.结果说明,构建携带LMP基因的转基因动物模型已成功.     

人SR-AI转基因小鼠遗传与繁育的研究

目的探讨被转入的人SR-AI基因的整合与复制情况及其对小鼠繁育的影响.方法采用类似系祖建系的方法,对人SR-AI转基因小鼠的2、3534、3560、3638、3639五个系列进行了繁育.并用PCR和Southermblot的方法,检测五个系列小鼠尾组织的DNA样品.结果五个系列小鼠共产仔431只,PCR检出阳性小鼠178只,阳性率为41.2%.在3639系的F1、F2、F3代及纯合子转基因鼠中PCR阳性率分别为47.8%、71.3%、75%和100%.结论人SR-AI基因在子代鼠中能稳定遗传,且对生殖和发育无明显影响.     

组织纤溶酶原激活剂突变体在转基因小鼠乳腺中的表达

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体——长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将它插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAtPA的转录、翻译受控于BLG基因的5′、3′序列,再将所构建的BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和Southern印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁中检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的9只F1代子鼠中,有5只是阳性的,tPA表达水平维持在1～2μg/ml,BLGtPA融合基因整合到小鼠基因组,能稳定地遗传给子代。     

胰腺组织表达Cre重组酶转基因小鼠的建立及鉴定

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性基因剔除研究的重要工具。为了建立胰腺组织特异性Cre转基因小鼠,我们通过PCR克隆了大鼠胰岛素基因启动子,并用它指导Cre基因在胰岛细胞中的特异性表达。在Cre重组酶基因5′端添加了真核核糖体结合序列和核定位序列以使Cre重组酶能穿越核膜在细胞核中发挥功能;同时,在Cre基因3′端添加了含内含子的3′端人生长激素基因。表达载体经显微注射导入小鼠受精卵以建立转基因小鼠。PCR检测显示共获得7只Cre整合阳性的转基因首建者小鼠;RTPCR结果表明其中1只首建者小鼠的子代鼠在胰腺中转录了外源基因,进一步的Southern杂交结果表明,该转基因小鼠能够在胰腺中表达有功能的Cre重组酶。      

转基因小鼠乳腺表达长效组织纤溶酶原激活剂

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将其插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAt-PA的转录、翻译受控于BLG基因的5′、3′序列,将此构建BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和DNA印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的F1代子鼠中,9只中有5只是阳性的,tPA表达水平维持在1-2μg/ml,说明BLG-tPA融合基因整合到小鼠基因组,能稳定地遗传给子代。为今后利用牛、羊作为生物反应器表达LAtPA奠定了基础。     

Transgenic expression of human LAMA5 suppresses murine Lama5 mRNA and laminin α5 protein deposition

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ～6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1-α5 isoform switch, or that for mouse laminin β1-β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.     

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice

Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25–30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.     

Conserved Regulatory Mechanisms of Tyrosinase Genes in Mice and Humans

In vertebrates, melanin production is restricted to pigment cells. This cell type-specific melanogenesis is considered to involve cell type-specific expression of the tyrosinase gene. Recently, there have been several reports that sequences in the 5’ flanking region of the mouse tyrosinase gene are responsible for cell type-specific expression of the transgene in mice. As the first step in the study of the evolution of the regulatory mechanisms for tyrosinase gene function in vertebrates, we constructed a fused gene, hg-Tyrs-J which includes a 1.0-kb 5’ flanking sequence of the human tyrosinase gene fused with mouse tyrosinase cDNA. By introducing the fused gene into fertilized eggs of albino mice, we obtained two mice that exhibited pigmentation in the skin and eyes and established a transgenic line from one of them. Further analyses revealed that the transgene was expressed cell type-specifically in these transgenic mice. We conclude, therefore, that the 1.0 kb 5’ upstream region of the human tyrosinase gene contains conserved cis-elements essential for cell type-specific expression of the tyrosinase genes in mice and humans. Results of our study may provide a clue to elucidate the evolutionary process of regulatory mechanisms of the tyrosinase gene.     

心脏特异表达Dhcr24转基因小鼠的建立和表型分析

目的建立心脏特异表达小鼠24-脱氢胆固醇还原酶基因（Dhcr24）转基因小鼠,研究该基因在心脏中表达对小鼠心脏发育,形态和功能维持中的作用。方法RT-PCR法克隆小鼠24-脱氢胆固醇还原酶基因,把Dhcr24基因插入-αMHC启动子下游,构建转基因表达载体,通过显微注射法建立Dhcr24 C57BL/6J转基因小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,RT-PCR和Western Blotting检测基因表达水平,光学显微镜和超声检测不同月龄Dhcr24转基因小鼠心脏的组织结构改变。结果建立了2个品系的心脏特异表达Dhcr24转基因小鼠。转入的Dhcr24基因在心脏组织的表达水平超过内源性Dhcr24的3倍。心脏组织学和超声检查证实：Dhcr24转基因小鼠的心室壁变厚,心腔变小,但心脏功能保持正常。结论成功建立了心脏特异表达Dhcr24转基因小鼠,Dhcr24基因在心脏组织的过度表达对小鼠心脏发育和功能维持中的作用需要进一步探讨。     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

系统表达HB-EGF转基因小鼠的建立

目的建立系统表达肝素结合性表皮生长因子（HB-EGF）的转基因动物模型,利用转基因动物模型研究HB-EGF与组织纤维化的关系。方法RT-PCR法克隆小鼠HB-EGF基因,将其插入Chickenβ-actin启动子下游,构建Chickenβ-actin-HB-EGF表达载体,利用显微注射的技术把表达载体注射到受精卵的雄原核中,建立HB-EGF转基因小鼠。利用特异引物PCR的方法鉴定转基因的基因型,采用Western Blot方法鉴定HB-EGF基因在全身组织的表达。分别取HB-EGF转基因鼠与同窝阴性小鼠的肝、肾、肺及膀胱组织进行Massion染色。结果建立了系统表达HB-EGF转基因小鼠,Western Blot发现其HB-EGF在肝、肺、肾及膀胱的表达与同窝阴性对照小鼠相比明显增加。Massion染色结果表明转基因鼠肝、肺、肾及膀胱组织纤维化程度明显高于同窝阴性对照小鼠。结论成功建立了系统表达HB-EGF转基因小鼠,HB-EGF的过度表可显著加重组织纤维化程度。     

Mouse models for methylmalonic aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.     

Cell type-specific expression of the human renin gene.

We have previously produced transgenic mice carrying the human renin gene, whose expression is regulated in a tissue-specific manner. In the present study, we further characterized expression of the transgene. Northern blot analysis showed that the human renin gene is expressed in the kidney but not in the liver of two lines of transgenic mice with 10 and 50 copies of the transgene, suggesting that the integrated copy number of the human renin gene does not influence the dominant-renal expression pattern. Immunohistochemical study using a monoclonal antibody specific for human renin demonstrated that expression of human renin in the transgenic mouse kidney is confined to the epithelioid juxtaglomerular cells. Transfection experiments indicated that the chloramphenicol acetyltransferase fusion gene containing the 3-kb upstream sequences of the renin gene is activated only in human epithelioid embryonic 293 cells derived from kidney but not in human HepG2 cells from liver. These findings suggest that transfer of the cloned renin gene into mice and in vitro cultured cell lines can give rise to cell type-specific expression.     

The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice.

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.