【作者】 卢宇；

【导师】 袁慧军；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉—头颈外科， 2011， 硕士

【摘要】 DFNA64是本课题组在一个常染色体显性遗传非综合征型耳聋家系中定位的新的耳聋基因,通过定位区域已知基因直接测序找到与耳聋表型共分离的DIABLO基因c.377C>T杂合突变。DIABLO是线粒体内膜上非常重要的细胞凋亡促进蛋白,本课题组研究提示p.S126L突变体对于外界刺激更加敏感,可引发线粒体膜电位慢性损伤。进一步的研究需要在动物模型中复制人类的临床表现,进而验证和分析突变型DIABLO对内耳毛细胞的损伤作用,从而揭示致病基因突变导致耳聋的机理、机制,为预防和治疗的可能性打下基础。一.DFNA64转基因小鼠模型的建立与鉴定本研究利用哺乳动物高效表达载体pcDNA3.1 (+)构建包含CMV启动子、人工构建内含子、目的基因cDNA编码序列、多聚腺苷酸信号等元件的转基因载体,通过原核DNA显微注射转基因技术将构建的外源转基因载体DNA片段注射到CDl远交系小鼠合子的前核中,分别建立并得到了整合有突变型和野生型Diablo基因序列的DFNA64转基因小鼠模型；利用PCR鉴定小鼠的基因型；原代转基因阳性小鼠自交建立多个转基因品系及纯合子转基因小鼠；real-time PCR检测Diablo基因在神经系统组织中的表达量,证实Diablo基因在DFNA64转基因小鼠体内的过量表达。二.DFNA64基因敲入小鼠模型的建立本研究利用定点同源重组原理和基于ES细胞的基因打靶技术建立DFNA64基因敲入小鼠模型,在129品系ES细胞基因组中置入与人类DFNA64突变同源的p.S124L突变,获得嵌合体小鼠。载体的设计采用一长一短两段目的基因的同源序列,同源序列之间插入突变序列和loxP序列锚定的正选择筛选的neo基因,将线性化的载体DNA电转至ES细胞,通过定点同源重组整合到ES细胞基因组序列,利用抗生素G418和长片段PCR联合筛选,剔除未整合和随机插入载体序列的ES细胞,筛选到阳性的ES细胞经显微注射进入宿主囊胚,体外培养后移植到同步化假孕小鼠,发育为嵌合体小鼠。DFNA64小鼠模型的建立,为DIABLO基因功能研究、致聋机制、发病前预防、药物筛选等后续研究工作提供平台。更多还原

【Abstract】 A new DFNA locus named DFNA64, was mapped in an autosomal dominant non-syndromic hearing loss family. By the means of direct sequencing of candidate genes in mapped region, heterozygous missense mutation c.377C>T was identified in DIABLO gene, which was segregated with the disease phenotype. DIABLO plays a key role in the promotion of apoptosis by binding the inhibitor of apoptosis protein. The previous investigations suggest mutant DIABLO while retaining the pro-apoptotic function, triggers significant degradation of both wildtype and mutant Smac/Diablo, rendering host mitochondria susceptible for calcium-induced membrane potential loss.. Mutant mouse models are necessary to understand the mechanism of inner ear hair cells damage by mutant DIABLO.1. Creation and identification of DFNA64 transgenic mouse modelIn this study, we obtained two lines of transgenic mouse models expressing wild and mutant diablo respectively by microinjection of linearized plasmid construct into fertilized eggs of CD1 strain mice. The construct is derived from pcDNA3.1(+), which is designed for high-level stable and transient expression in mammalian hosts. It contains CMV promotor, mutant or wild cDNA fragment of diablo, artifical intron and bGH-polyA signal. After identified the genotype of founders, generate transgenic mouse by cross the founders. Real-time PCR identified the overexpression of both wild and mutant diablo in nervous system.2.Creation of DFNA64 knock-in mouse modelWe also established knock-in mouse model by gene targeting. Targeting ES cells from 129 strain mice by electro-transformation, with construct consisting of two homologous sequences with floxed neo castle and mutation inserted. The targeted ES cells were screened out by antibiotic G418 and PCR reaction, then microinjected into embryos. We obtained five chimera mice. DFNA64 mouse models play significant roles in the study into gene function of DIABLO, mechanism of hearing loss, prevention before onset, therapeutic approaches.更多还原