Prestin基因相关人耳聋疾病动物模型的建立

发布时间：2018-07-01 18:49

本文选题：Prestin + 基因敲入　；参考：《山东大学》2011年硕士论文

【摘要】：哺乳动物的听力极为敏感,这是由于耳蜗外毛细胞(Outer Hair Cells, OHC)中有一种生物放大器。这种放大器通过外毛细胞胞体长度的改变将基底膜的振动放大100倍,从而使人听到声音。外毛细胞的胞体长度的改变是由某种电动蛋白驱动的,多年来人们一直在寻找能够引起外毛细胞胞体振荡的电动蛋白。在2000年Prestin基因被克隆后,研究者利用基因敲除技术证明了Prestin就是内耳外毛细胞中的电动蛋白。Prestin是听力放大的核心基因,它与人类耳聋疾病密切相关,至今已发现两种引起入耳聋的Prestin点突变,但目前对Prestin突变的分子病理机制知之甚少。关于Prestin在人类中的突变致聋已有文献报道：2003年在第2内含子和第3外显子(ATG)交汇处一种点突变(IVS2-2A/G)被发现,它处于Prestin mRNA剪切相关的区域；2007年在第6外显子中,发现了另一个点突变(R150Q)。这两种点突变均能引起人听力的损失。鉴于人类耳聋是发病率相对较高的一种疾病,利用制作相应疾病动物模型的方法研究治病机理具有极高的价值。我们利用基因敲入的方法,制作Prestin基因敲入小鼠模型,以期精确模拟导致人类耳聋疾病的Prestin基因第6号外显子上的点突变(R150Q)。我们可以通过对这种小鼠模型的表型分析和研究,避免了在人体实验材料以及实验技术不可操作性的限制。本研究选择对第6外显子进行特定的点突变(R150Q),原因是利用它不仅可以发现Prestin突变致聋的分子病理机制,还可以在Prestin功能的基础研究上有新的突破,因为Prestin蛋白在耳蜗放大功能中的基础研究虽然取得了很大进展,但也存在许多尚未解决的重要问题,我们准备制作的小鼠模型将在解决这些问题中起到关键作用。实验方法：第一,我们设计并构建了基因敲入的打靶载体；第二,通过电转化的方式,把线性化好的载体电转化到胚胎干细胞(Embryonic Stem cells, ES)中,用含有G418的培养基筛选培养ES细胞,细胞长到一定的数量时,收集细胞；第三,提取细胞的基因组DNA,用先前设计好的筛选引物进行PCR扩增筛选,如果同源重组成功,PCR就可以扩增出特异的条带；第四,我们把经过筛选得到的ES细胞通过显微注射的方式,注射到小鼠囊胚期的胚胎中,最终获得嵌合体小鼠；第五,通过杂交来确定是否具有生殖系转移；最后,如果得到了具有生殖系转移的小鼠,通过杂交和自交得到纯和体后代,进行生理、生化、蛋白等方面的分析。结果：成功的制作了Prestin Konck-in载体,转染到ES中。通过筛选得到了发生正确同源重组的克隆,并通过显微操作技术将得到的ES细胞注射到囊胚,最终获得高嵌合率的小鼠。本研究的工作为进一步阐明外毛细胞中听觉发生的分子机制奠定了基础。
[Abstract]:The hearing of mammals is very sensitive. This is due to a biological amplifier in the Outer Hair Cells (OHC). This amplifier amplifies the vibration of the basement membrane by 100 times that of the cell body length of the outer hair cell, thus making people hear the sound. The changes in the length of the cell body of the outer hair cells are driven by a certain electroprotein. After the Prestin gene was cloned in 2000, the researchers used gene knockout technique to prove that Prestin is the core gene of the hearing amplification, which is closely related to the deafness disease of human beings. It has been developed so far. There are two Prestin point mutations that cause deafness, but little is known about the molecular pathogenesis of Prestin mutations.
The mutation induced deafness of Prestin in humans has been reported in the literature: a point mutation (IVS2-2A/G) was found at the intersection of second introns and third exons (ATG) in 2003. It was found in the region of Prestin mRNA shear related; in 2007, another point mutation (R150Q) was found in exon Sixth. These two point mutations all cause human hearing. Loss.
In view of human deafness is a relatively high incidence of disease, it is of great value to use the method of making the animal model of the corresponding disease to study the mechanism of the disease. We use the method of gene knock to make the Prestin gene into the mouse model in order to accurately simulate the point on the exon sixth of the Prestin gene that leads to the deafness of human beings. Mutation (R150Q). We can avoid the limitation on the non operability of experimental materials and experimental techniques by analyzing and studying the phenotype of this mouse model. This study chooses a specific point mutation (R150Q) for sixth exons, because it can be used not only to find the molecular pathological mechanism of the deafness caused by Prestin mutation but also to find the molecular mechanism of the deafness. There are new breakthroughs in the basic research on Prestin function, because the basic research of the Prestin protein in the cochlear enlargement has made great progress, but there are many important problems that have not been solved. The mouse model we are preparing to produce will play a key role in solving these problems.
Experimental methods: first, we designed and constructed the targeting vector of gene knocking; second, through the electrical transformation, the linearized carrier was converted into Embryonic Stem cells (ES), and ES cells were screened and cultured with G418 medium, and the cells were collected when the cell was long to a certain number; and third, extract fine. The genomic DNA of the cell is screened by PCR amplification with previously designed primers. If the homologous recombination is successful, the specific bands can be amplified by PCR; fourth, we inject the selected ES cells into the mouse blastocyst embryos by microinjection, and finally get the chimerism mice; fifth, through hybridization. To determine whether or not a reproductive system is transferred; finally, if a mouse with a reproductive system has been transferred, the analysis of physiological, biochemical, protein, and other aspects is carried out by cross and self crossbreeding of pure and body offspring.
Results: the Prestin Konck-in vector was successfully produced and transfected into ES. The correct homologous recombination was obtained by screening, and the ES cells were injected into the blastocyst by micromanipulation, and the mice with high chimerism were finally obtained. The work of this study is to further clarify the molecular mechanism of auditory occurrence in the outer hair cells. Set the foundation.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764;R-332

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