共查询到20条相似文献，搜索用时 31 毫秒
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【目的】构建针对斑马鱼Danio rerio卵细胞的可诱导消融品系,并了解其卵巢组织在条件诱导下的去除规律和特性,为相关领域的应用提供材料基础。【方法】利用Tol2转座系统建立卵细胞特异表达硝基还原酶(Nitroreductase,NTR)的转基因斑马鱼品系;通过定期采样,连续观察、解剖、组织切片等方法研究甲硝唑(Metronidazole,Mtz)对成熟雌鱼外观及卵巢结构的影响;冰冻切片结合TUNEL检测,了解条件性诱导对卵巢细胞凋亡的影响。【结果】获得囊胚细胞具特异荧光标记的转基因鱼,并形成稳定遗传品系。经Mtz诱导,该品系成熟雌鱼卵巢结构逐步退化、萎缩,卵母细胞渐渐消融,提示NTR在卵细胞特异表达;检测发现Mtz可导致卵细胞的凋亡,推测卵巢的组织变化为细胞凋亡所致;撤销Mtz胁迫卵巢可再生并恢复生育功能,说明卵巢的消融过程是可逆的。【结论】获得的转基因斑马鱼可通过条件诱导实现卵巢消融与再生。 相似文献
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Schlabach MR Luo J Solimini NL Hu G Xu Q Li MZ Zhao Z Smogorzewska A Sowa ME Ang XL Westbrook TF Liang AC Chang K Hackett JA Harper JW Hannon GJ Elledge SJ 《Science (New York, N.Y.)》2008,319(5863):620-624
Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells. 相似文献
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S Kamel-Reid M Letarte C Sirard M Doedens T Grunberger G Fulop M H Freedman R A Phillips J E Dick 《Science (New York, N.Y.)》1989,246(4937):1597-1600
A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies. 相似文献
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The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease. 相似文献
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Lecuit M Vandormael-Pournin S Lefort J Huerre M Gounon P Dupuy C Babinet C Cossart P 《Science (New York, N.Y.)》2001,292(5522):1722-1725
Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated. 相似文献
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莱茵衣藻细胞核转化系统研究进展 总被引：1，自引：0，他引：1
莱茵衣藻(Chlamydomonas reinhardti)是一种3套基因组都能进行遗传转化的真核生物.随着转基因技术的不断完善,利用衣藻作为受体生物进行外源基因表达的研究已经成为新的研究热点.特别是利用衣藻核基因组转化系统表达外源基因的研究也取得了一系列的进展.在受体衣藻品系的选择、遗传转化效率、表达调控等方面进行的系列研究,为建立一套稳定高效的外源基因表达系统打下了坚实的基础.文章对近几年在衣藻细胞核遗传转化系统等方面的研究进展进行了综述. 相似文献
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M J Dycaico S G Grant K Felts W S Nichols S A Geller J H Hager A J Pollard S W Kohler H P Short F R Jirik 《Science (New York, N.Y.)》1988,242(4884):1409-1412
Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of the human alpha 1-antitrypsin (alpha 1-Pi) gene. All of the alpha 1-Pi transgenic mice expressed the human protein in the liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The human M-allele protein was secreted normally into the serum. However, the human Z-allele protein accumulated in several cell types, but particularly in hepatocytes, and was found in serum in tenfold lower concentrations than the M-allele protein. Mice in one lineage carrying the mutant Z allele expressed high levels of human alpha 1-Pi RNA and displayed significant runting (50% of normal weight) in the neonatal period. This lineage was found to have alpha 1-Pi-induced liver pathology in the neonatal period, concomitant with the accumulation of human Z protein in diastase-resistant cytoplasmic globules that could be revealed in the Periodic acid-Schiff reaction (PAS). The phenotype of mice in the strain expressing high levels of the Z allele is remarkably similar to human neonatal hepatitis, and this strain may prove to be a useful animal model for studying this disease. 相似文献
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鱼类侧线系统源自胚胎期的侧线基板,侧线基板的特化受到多种信号的综合作用。利用转基因斑马鱼hsp:wnt8a-egfp和hsp:dkk1-egfp,增强或抑制Wnt信号,通过YO-PRO-1染色和毛细胞计数,研究斑马鱼中Wnt信号对侧线系统发育的影响。热激诱导Dkk1过表达后,斑马鱼侧线系统的神经丘不能形成。碱性磷酸酶染色和整体原位杂交结果表明:Wnt信号是神经丘毛细胞前体细胞以及侧线基板标记基因Eya1和Six2b表达所必需的。综上结果:Wnt信号可能通过调控侧线基板形成影响斑马鱼的侧线发育。 相似文献
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【目的】构建转入靶向猪Jiv基因shRNA干扰片段的阳性细胞株,通过比较各细胞株对猪瘟病毒增殖的干扰效果,筛选对猪瘟病毒增殖有明显抑制作用的细胞株,为抗猪瘟转基因猪的构建提供材料。【方法】研究设计了靶向猪Jiv基因的4个shRNA干扰片段,并构建插入干扰片段的慢病毒(P1、P2、P3、P4)。将慢病毒分别转染PK-15细胞,阳性细胞接种猪瘟病毒后72 h用实时荧光定量PCR检测猪瘟病毒RNA的量,以比较4种干扰细胞株对猪瘟病毒增殖的干扰效果。将对猪瘟病毒增殖有较好干扰效果的P2慢病毒干扰载体转染猪胎儿成纤维细胞,获得稳定表达靶向猪Jiv基因shRNA干扰片段的猪胎儿成纤维细胞株,作为抗猪瘟病毒转基因猪构建的供体细胞,将细胞核移植到成熟的去核猪卵母细胞中,获得体细胞核移植胚胎,移植入受体母猪输卵管中进行转基因猪构建,对获得的转基因猪进行外源基因的鉴定。【结果】获得了4株转入靶向猪Jiv基因shRNA干扰片段的PK-15细胞株,其中转入P2干扰载体的细胞株对猪瘟病毒的增殖有明显的抑制作用,将转入P2干扰载体的猪胎儿成纤维细胞株为核供体细胞通过体细胞核移植,获得经鉴定为外源基因插入阳性的转基因猪。【结论】细胞Jiv基因的表达对猪瘟病毒的增殖有一定的影响,筛选获得的一个干扰细胞株对猪瘟病毒的增殖有明显的干扰效果;通过体细胞核移植技术获得一头转入靶向猪Jiv基因shRNA干扰片段（P2）的转基因猪。 相似文献
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Thymic requirement for clonal deletion during T cell development 总被引：14，自引：0，他引：14
During T cell differentiation, self tolerance is established in part by the deletion of self-reactive T cells within the thymus (negative selection). The presence of T cell receptor (TCR)-alpha beta + T cells in older athymic (nu/nu) mice indicates that some T cells can also mature without thymic influence. Therefore, to determine whether the thymus is required for negative selection, TCR V beta expression was compared in athymic nu/nu mice and their congenic normal littermates. T cells expressing V beta 3 proteins are specific for minor lymphocyte stimulatory (Mlsc) determinants and are deleted intrathymically due to self tolerance in Mlsc+ mouse strains. Here it is shown that V beta 3+ T cells are deleted in Mlsc+ BALB/c nu/+ mice, but not in their BALB/c nu/nu littermates. Thus, the thymus is required for clonal deletion during T cell development. 相似文献
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为了探讨核定位信号肽S413-PV和转运肽Tp10这2种融合肽对转基因效率的影响,将S413-PV和Tp10与线性化质粒分别以质量比50∶1、100∶1、200∶1混合制成转染液,注射斑马鱼单细胞期胚胎,分别检测转染24 h、120 h后胚胎的阳性率和成活率。结果表明:与对照组相比,转染24 h后,S413-PV组胚胎阳性率极显著上升,胚胎成活率均下降,但差异不显著;Tp10组阳性率均呈上升趋势,200∶1组差异极显著,其他组差异不显著,胚胎成活率均显著下降;转染120 h后,S413-PV组胚胎阳性率均极显著上升,胚胎成活率均呈下降趋势,100∶1、200∶1组差异极显著;Tp10组胚胎阳性率上升,200∶1组差异显著,胚胎成活率均极显著下降。以上结果表明,S413-PV可显著提高斑马鱼转基因效率,对胚胎毒性较小;Tp10对转基因效率影响不显著,且对胚胎毒性较强。 相似文献
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Thymus adenylate cyclase activity during murine leukemogenesis 总被引：2，自引：0，他引：2
Activity of thymus adenylate cyclase was more than three times higher in leukemic AKR mice than in nonleukemic AKR mice and CBA mice. Preleukemic AKR mice that had no evidence of leukemia but were expected to soon develop the disease exhibited similarly elevated activities of thymus adenylate cyclase. 相似文献
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蜡梅凝集素基因克隆及其对蚜虫、蛞蝓抗性分析 总被引：1，自引：0，他引：1
【目的】克隆蜡梅凝集素基因并研究其抗蚜虫和抗蛞蝓活性,为植物抗虫基因工程提供理论依据和试验材料。【方法】通过随机挑选克隆测序的方法,从蜡梅花cDNA文库中克隆蜡梅凝集素基因,并构建植物表达载体,用农杆菌介导的叶盘法转化烟草,并对转基因植株进行抗蚜和抗蛞蝓试验。【结果】从蜡梅花cDNA文库中克隆到了一个凝集素基因CpLEC(GenBank登录号：DQ352145),该基因全长871 bp, ORF框长558 bp, 编码185个氨基酸。该基因DNA分析表明其不含内含子。具有单子叶植物甘露糖凝集素基因家族的特征,有2个典型的甘露糖结合位点(QXDXNXVXY),和1个变异的可能结合位点(HXGXNXVXY)。Southern杂交表明,该基因在蜡梅基因组中为多拷贝。构建了35S启动子控制下的CpLEC基因的植物表达载体pBI121-CpLEC,利用根癌农杆菌介导法转化烟草获得抗卡那霉素的植株。PCR和半定量RT-PCR分析表明,CpLEC基因已经整合到植物基因组中并在转录水平上得到了有效表达。抗蚜虫鉴定表明,转基因植株及其离体叶片有效地抑制了蚜虫的群体增长率,平均抑制率分别为60%和68%。抗蛞蝓试验结果表明转基因植株有明显的抗蛞蝓活性,转基因烟草虫害指数为0.17,远低于非转基因烟草的虫害指数0.96。【结论】克隆获得了蜡梅凝集素基因CpLEC,通过转基因手段分析其抗蚜和抗蛞蝓活性,结果表明该基因对抗虫基因工程有重要的应用价值。 相似文献
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多抗PVY、TMV和CMV转基因烟草的培育 总被引：1，自引：0，他引：1
【目的】利用RNA介导抗性培育抗多种植物病毒的转基因烟草。【方法】分别以马铃薯Y病毒（PVY）、烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）全长衣壳蛋白（CP）基因为模板,通过设计PCR引物和亚克隆获得PVY CP 3′端长度100 bp、TMV CP 3′端长度100 bp和CMV CP 3′端长度200 bp的cDNA片段并拼接成嵌合基因,并以此为模板构建反向重复结构嵌合基因的植物表达载体pRHPTC。将pRHPTC通过冻融法导入农杆菌LBA4404,采用叶盘法转化烟草NC89,然后测定转基因烟草对3种病毒的抗性。【结果】经卡那霉素筛选和PCR检测,共获得276株转基因烟草。Southern和Northern blot分析表明,外源基因以不同拷贝数整合于烟草基因组中;不同转基因植株中病毒RNA的积累量存在显著差异。抗病性检测显示：23%左右的转基因植株表现出对3种病毒侵染的抗性。对转基因植株扩繁后代和T1代的抗性分析表明：多病毒抗性表现稳定。【结论】利用RNA介导的抗病毒基因工程可获得同时抗多种病毒的转基因烟草,其抗病性在T0代扩繁植株和T1代植株中得到稳定遗传。 相似文献
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试验以仙客来热激蛋白基因HSP21.4为材料,经PCR扩增的方法在目的片断两端添加SacⅠ位点,然后与植物表达载体pBI121连接,转化感受态大肠杆菌,经PCR和SacⅠ及XbaⅠ酶切验证,确定已将该热激蛋白基因连接到植物表达载体上,将重组质粒命名为pBI-CpHSP21.4,并采用冻融法完成了对pBI-CpHSP21.4质粒的农杆菌转化,为验证该热激蛋白基因的功能及转基因植物表达奠定基础。 相似文献
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A regulatory element consisting of a MAR element sequence of Drosophila heat shock genes and enhancer from a long terminal repeat of the mouse mammary tumor virus is created for studying the possibility
of increasing the expression level of hybrid genes in transgenic animals. Two lines of transgenic mice containing the described
regulatory element and hybrid gene intended for expressing human beta interferon in milk of transgenic animals are obtained
by the method of coinjection into the pronuclei of murine zygotes. It is shown that the use of the given regulatory element
doesn’t lead to an increase of activity of human beta interferon in milk compared with transgenic rabbits containing only
the hybrid gene for expressing beta interferon. Furthermore, a decrease of tissue-specific expression of beta interferon is
observed with the use of the regulatory element. 相似文献
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Anderson MS Venanzi ES Klein L Chen Z Berzins SP Turley SJ von Boehmer H Bronson R Dierich A Benoist C Mathis D 《Science (New York, N.Y.)》2002,298(5597):1395-1401