【作者】 孙汉堂；

【导师】 肖明振； 费俭；

【作者基本信息】 中国人民解放军第四军医大学 ， 口腔临床医学， 2003， 博士

【摘要】 牙本质涎磷蛋白(dentin sialophosphoprotein，DSPP)包括牙本质涎蛋白(dentin sialoprotein，DSP)和牙本质磷蛋白(dentin phosphoprotein，DPP)，属于非胶原蛋白，因最早被发现是由成牙本质细胞合成和分泌，一度被认为是牙本质的特异性蛋白而得名。近年来的研究表明，DSPP在其他组织和器官(如骨、软骨、内耳等)中也表达，只是表达水平相对较低而已，提示DSPP的功能可能并非局限于牙齿的发育和矿化。为进一步探讨DSPP在牙齿发育和矿化中的作用，并在此基础上对DSPP的功能做更加全面的研究，本研究在DSPP功能研究的在体模型的构建方面做了一些尝试，核心内容是建立DSPP的转基因和基因敲除小鼠系。本文是全面研究规划中的一部分，即DSP转基因小鼠模型、DSPP四环素调控性转基因小鼠体系、DSPP特异性启动子调控LacZ转基因小鼠模型的建立和DSPP基因敲除载体的构建以及胚胎干细胞基因打靶。 一、DSP转基因小鼠模型的构建 通过亚克隆将pcDNA3.1中的CMV启动子替换为cβ-actin启动子，构建载体pcDNA3.1-CX，然后将PCR获得的、末端带有HA-Tag的DSP基因编码序列克隆到pcDNA3.1-CX中，构建DSP转基因载体pcDNA3.1-CX-DSP；用显微注射的方法将BglII酶切线性化的载体DNA注射到小鼠受精卵的雄 第四军医大学博士学位论文原核，移植到假孕母鼠的输卵管；仔鼠出生后，用PCR、基因组Southern杂交检测阳性小鼠，结果获得4只GO代小鼠（founder）；阳性鼠分别传代开始建系。选其中一个单系进行了初步的分析，RTPCR显示转基因能在多个器官中表达。h、DSPP四环素调控性转基因小鼠体系的构建 将pTetonNSE载体中的NSE启动子替换为启动于c p仓ctin，构建调控载体pTeton（X；将DSPP基因编码序列克隆到pTREZ中，构建受控载体pTREZ＊SPP通过显微注射将线性化载体导入小鼠受精卵，制备GO代小鼠，经PCR、基因组Soutnern杂交检坝卜性小鼠，pTeton－CX手pTREZoSPP分别获 11只、10只GO代小鼠。阳性鼠分别传代开始建系。选pTet｛n（X转基因小鼠一个单系进行了初步的分析，RTPCR结果表明小鼠多种器官中均可表达转录因子tTA。三、DSPP基因特异性启动子的克隆和pTNlPM工acZ转基因小鼠的构建 据文献报道，在软件DNAstar辅助下设计一对引物，以小鼠基因组DNA为模板，PCR获得约1石 kb的片段，序列测定证实为DSPP基因特异性启动子；通过亚克隆构建了特异性启动子一LacZ转基因载体，命名为pTN－DPM工acZ，显微注射法制备pTN0PM＊acZ转基因小鼠。经PCR筛选，共获得 12只GO代小鼠。四、DSPP基因敲除载体的构建和ES细胞基因打靶的尝试 根据NCBI GenBank提供的序列，软件辅助设计并合成上、下游两条同源臂的两对引物：以用细胞基因组＊＊A为模板，P＊R获得上、下游两条同源臂；将同源臂测序确认后，和筛选标志按一定顺序克隆到载体pBluescript中，构建了G418筛选的们打靶载体和G418／GANC筛选的＃2打靶载体；酶切和序列测定确认构建无误后，N。ti酶切使载体线性化用于ES 3 第四军医大学博士学位论文细胞基因打靶。共进行了 3次打靶，鉴定了 3 74个细胞克隆。虽然没有获得阳性克隆，但得到了一定的经验，为以后的研究打下了基础。 DSP转基因小鼠模型、DSPP四环素调控性转基因小鼠体系、DSPP特异性启动子一LacZ转基因小鼠模型的建立以及DSPP基因敲除载体的构建和ES细胞基因打靶的尝试，为全面研究DSPP的功能奠定了基础。更多还原

【Abstract】 Dentin sialophosphoprotein(DSPP), which was considered to be cleaved into dentin sialoprotein(DSP) and dentin phosphoprotein (DPP) at posttranscriptional level, belongs to non-collagenous protein(NCP), and got its name by the fact that it was first discovered to be secreted by odontoblasts and appear at the mineralization front of dentine. It was once regarded as the unique dentine-specific protein, but according to recent studies, it was also expressed in other tissues and organs, such as bone, cartilage, and even inner ear, which means that perhaps it was not a dentine-specific protein, and it might have other functions rather than involving in the mineralization process of tooth. To further the studies about the role of DSPP in the development and mineralization of tooth, and start the researches about the functions of DSPP in other tissues and organs, attempts of the generation of in vivo biomodel organism for the function analysis of DSPP were carried out. The most important task of the plan is to establish DSPP transgenic and knockout mice models. The followings were parts of this plan, including the generation of DSP transgenic mice, DSPP tetracycline-responsive transgenic mice system, the construction of DSPP knockout construct and the attempt on DSPP knockout by ES cell gene targeting.1. Generation of DSP transgenic micepcDNA3.1 -CX was constructed by substituting CMV promoter of pcDNA3.l with c β -actin promoter, and the ultimate transgenic construct was established by cloning DSP coding sequence(with a HA-Tag linked to its C-terminal) into pcDNA3.1-CX. The transgenic DNA was linearized by BgIII digestion and then microinjected into the male pronucleus of the mouse zycotes. The tail DNA of pups was tested by PCR and Southern blot. 4 founders were characterized and breeding process was carried out. RT-PCR showed that the transgene was expressed in many organs.2. Generation of tetracycline-responsive transgenic mice system of DSPPThe promoter of pTet-on-NSE was replaced by c β -actin promoter, which led to the construction of the tetracycline- responsive vector pTet-on-CX; and the DSPP transgenic vector pTRE2-DSPP, was constructed by subcloning DSPP coding sequence from pBlueScript-DSPP to pTRE2. Candidates of the transgenic mice were generated by microinjection and pups were screened by PCR and Southern blot. All together 11 and 10 founders were got, respectively; and breeding process was carried out. RT-PCR showed that the transgene pTet-on-CX could be expressed in many organs.3.Cloning of DSPP specific promoter and generation of DSPP-specific -promoter-LacZ transgenic mice.According to the sequence reported by MacDougall et al, primers for DSPP specific promoter were designed, and PCR was performed. A 1.6 kb fragment was obtained and it was confirmed to be DSPP specificpromoter by sequencing. DSPP-specific-promoter-LacZ transgenic construct was constructed by subcloning, and transgenic mice were produced bymicroinjection and screened by PCR. 12 founders were obtained.4. Attempts on DSPP knockout by ES cell gene targetingThe primers were designed by the use of DNAstar software and were in accordance with the sequence provided by NCBI GenBank. Using genomic DNA of ES cells as template, PCR was performed to get the upper and lower homology arms of the targeting constructs. After characteration by sequencing, the homology arms were subcloned, together with the screening marker, e.g. hsv-tk-neo and PGK-TK, into pBluescript in a definite order. The ultimate constructs were confirmed by enzyme digestion and sequencing, and were linearized by NotI for the use of electrotransformation of the ES cells. Altogether 2 targeting constructs were constructed, 3 transformation experiments were performed and 374 ES cell clones were tested by PCR and Southern blot, no positive clone was found up to date.The generation of DSP and DSPP transgenic mice, and attempts on DSPP gene knockout make basis for further function analysis of DSPP.更多还原