【作者】 张鸿声；

【导师】 关雄； 胡宝成；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 条件性基因打靶是在常规基因打靶的基础上,结合重组酶介导的位点特异性重组技术发展而来的一种基因打靶技术。它可以将对特定基因的修饰限制于特定类型的细胞、组织甚至小鼠发育的特定阶段。基于Cre/LoxP系统的条件基因打靶,通常借助同源重组将两个LoxP序列置于靶基因重要功能域两侧的内含子中,希望获得表型正常的条件基因打靶小鼠;而后将该小鼠与细胞或组织特异性表达Cre重组酶的转基因小鼠杂交后,可望获得组织特异性基因剔除小鼠。其中,Cre重组酶只在特定的细胞或组织中表达,并且Cre/LoxP位点依赖的重组会将该细胞中的靶基因剔除,而其它类型细胞中由于没有Cre重组酶的表达,靶基因不会被删除而仍然保持原有的表达模式。这样,就为我们研究某些删除后导致胚胎期致死的基因功能提供了很好的研究工具。NPM是一种多功能蛋白,主要位于核仁区,能穿梭于核仁、核质和胞质之间,参与核糖体前体运输和合成及中心体的复制等过程,从而调控细胞的周期进程与生物机体发育。人类NPM基因位于染色体5q35,全长约23Kb,包含12个外显子。整个蛋白包含三个功能域:寡聚化分子伴侣功能域、组蛋白结合功能域和核酸结合功能域,并且具有一系列的定位信号序列(如核输出信号、核定位信号以及核仁定位信号等)及磷酸化位点。研究结果表明,在血液肿瘤中NPM经常发生突变,暗示它可能是一种肿瘤抑制基因。也有研究发现它在另外一些肿瘤细胞(如胃癌、结肠癌、卵巢癌、前列腺癌和肝癌)中过表达,也暗示NPM可能是一种致癌基因。究竟NPM在这些肿瘤发生发展过程中发挥了怎么样的作用还有待进一步的研究。鉴于NPM完全基因剔除小鼠的胚胎在发育中期死亡。为了进一步研究NPM在成体各组织器官及肿瘤发生发展中的生物学功能,我们试图建立NPM条件基因剔除小鼠模型,而建立小鼠模型的首要任务就是构建NPM条件基因剔除载体,基于NPM的N端和中间区域的重要性以及实验操作的可行性,我们将两个LoxP序列分别置于NPM基因组序列N端和中间功能域两侧内含子中。首先,我们从小鼠基因组BAC质粒中克隆了一段包含NPM全部基因组序列在内的13Kb的片段,连接至pBS KS [pBluescript II KS(+)]载体上,命名为pBSKS-NPM。然后我们再次从该段序列上克隆了大小为8 kb和1.8 kb的两条片段,分别作为NPM条件基因打靶载体的5’和3’同源臂,也分别连接到pBS KS载体上,选取插入方向正确的克隆。之后再将3’同源臂克隆到pGEX-KG载体上,并在此载体上通过Kpn I单酶切位点在第6个内含子处插入LoxP3。最后把两条同源臂分别克隆至通用打靶载体pLoxP I上,形成NPM基因第2-6个外显子被LoxP序列锚定的条件基因打靶载体,命名为pLoxP-NPM。PCR筛选、酶切鉴定和序列测定结果证明NPM条件基因打靶载体与预期结构一致。最后,将打靶载体Not I线性化后电击转染ES细胞,在含G418和Gancyclovir的选择性培养基中筛选7天后挑取抗性克隆53个。经PCR初步鉴定21个克隆为阳性克隆,对这些阳性克隆需进一步进行扩增后提取基因组DNA用Hind III酶切,用打靶载体的3’端外侧探针进行Southern blot,野生型将出现13kb条带,而NPM中靶等位基因由于插入NPM内含子中LoxP序列携带一个Hind III位点,中靶细胞将出现13kb和7232bp两条带。中靶ES细胞的成功获得将为进一步建立NPM组织特异性剔除小鼠模型奠定了基础。更多还原

【Abstract】 Conditional gene knockout is developed as being a new strategy with combination of site-specific recombination mediated by recombinase on the basis of tranditional gene targeting, in which modification on specific gene was confined in certain kind of cell, tissue even in special development period of mice. In conditional gene targeting system with Cre/LoxP recombination, the important functional domains of targeting gene were floxed by two LoxP sites, usually being inserted into some introns. And therefore Conditional knockout mice show normal phenotype. After mating with mice with special Cre expressoin in some cells or tissue, target gene will be deleted by Cre/LoxP recombination in the specific region. The gene will be kept intact expression in other tissues. So it is one good tool to study the function of some genes with deletion-lethality during embryo development.NPM (nucleophosmin),one of the multifunctional phosphoproteins, mainly locates in nucleoli and shuttles rapidly between the nucleus and cytoplasm. It can involve in transporting ribosomal precursor and centrosome duplication to regulate cell cycle process and organism development. In human NPM was mapped on 5q35. It contains 12 exons with the size of about 23 kb. From the N terminus, this protein contains one hydrophobic segment that is involved in oligomerization as well as chaperone activity, followed by two acidic stretches that are important for binding to histones, the remains and C-terminal domain which contains basic regions involved in nucleic-acid binding. NPM also contains nuclear localization signal, a nuclear export signal and nucleolar localization signal and multiphosphorylation sites.NPM was implicated in human tumorigenesis. NPM was frequently overexpressed in solid tumours of a diverse histological origin, such as gastric, colon, ovarian ,prostate and liver carcinomas, and genetic alterations occured frequently in haematopoietic tumours. It seems to function as either an oncogene or a tumour suppressor. Further investigation can address that what role of NPM is playing during tumor genesis and development. Target disruption of NPM gene resulted in embryonic lethality at mid-gestation. To study the function of NPM in vertebrate organgenesis and tumorigenesis, The model of NPM conditional gene knockout mice is very important. The first step was to make the conditional gene targeting vector. In the constructor, 2-6 exons of NPM were floxed by two LoxP sites.A 13kb genomic DNA fragment containing NPM full length gene, obtained from BAC by subcloning, was inserted into pBS KS [pBluescript II KS(+)] vector and named pBS KS-NPM. A 1.8kb DNA fragment containing exon 1, obtained from pBS KS-NPM by subcloning, was inserted into pBS KS vector which was used as the 5’homologous arm. A 8kb DNA fragment containing exon 2-9, obtained from pBS KS-NPM by subcloning as well, was inserted into pBS KS vector which was used as the 3’homologous arm. Then 3’homologous arm was inserted into pGEX-KG vector and named pGEX-3’arm to obtain suitable enzyme sites, and the LoxP3 was inserted into pGEX-3’arm at Kpn I restriction enzyme site within intron 6, this fragment was used as the final 3’homologous arm. Afterward 5’homologous arm and 3’homologous arm were cloned into classical targeting vector pLoxP I, respectively. Finally NPM conditional gene targeting vector were obtained, in which exons 2-6 were anchored by LoxP. The targeting vector was confirmed by PCR, restriction enzyme digestions and partial sequencing analysis, named pLoxP-NPM.Linearized by Not I targeting construction was introuduced into ES cells by electroporation. 53 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The clonies were amplified and subjected to genomic DNA preparation. And 21 clonies were confirmed by PCR identification. The genomic DNA were digested with Hind III and used for Southern blot analysis. A probe outside the 3’homologous arm was used for hybridization.13 kb positive band was found in wild type.The band was shift down from 13kb to 7232bp in NPM conditional targeted allete because a new Hind III site was introduced into the intron along with the LoxP site. Targeted ES cells are the basis to generate NPM conditional gene knockout mice.更多还原