骨骼肌特异性报告基因逆转录病毒载体的构建与验证
发布时间：2018-11-05 10:14
【摘要】： 目的：卫星细胞(muscle satellite cells)认为是保留在成年骨骼肌内具有增殖分化能力的肌源性细胞。建立简便快速的鉴定和筛选骨骼肌卫星细胞的方法对于卫星细胞培养、研究和应用都具有实际的意义。Desmin是目前知道的表达开始于骨骼肌卫星细胞并在肌纤维中高表达的标志蛋白。因此以Desmin基因的启动子元件控制的报告基因可望建立鉴定和筛选骨骼肌卫星细胞的简便方法。本课题目的是构建逆转录病毒报告基因载体，特异性表达于骨骼肌细胞中，并通过检测其绿色荧光蛋白表达，验证其在骨骼肌细胞中表达的特异性。并在此基础上将本实验所构建重组载体制备成逆转录病毒悬液，检测其在分化的P19细胞中的表达。 方法：(1)重组载体的构建与鉴定，首先用PCR法扩增所需要的目的片段，绿色荧光蛋白和新霉素基因(GFP+NEO)和desmin启动子和增强子(pro+enh)，然后利用限制性内切酶和T4 DNA连接酶，将目的基因连接到经NcoⅠ，BamHⅠ，EcoRⅠ和SalⅠ酶切后回收的逆转录病毒载体psuper线性片段上。(2)重组载体的骨骼肌卫星细胞表达特异性验证：将psuper和重组载体分别转染CHO和C2C12细胞，，24h后，倒置荧光显微镜下观察绿色荧光蛋白的表达，采集图片。(3)逆转录病毒悬液制备及其在分化P19细胞中的应用，逆转录病毒悬液转染C2C12和DMSO诱导分化的P19细胞，倒置荧光显微镜下观察绿色荧光蛋白的表达，采集图片。 结果：(1)通过对构建过程的每一步进行电泳分析，证明了所构建重组载体是正确的。(2)重组载体和psuper转染CHO和C2C12细胞后，在C2C12细胞表达水平相当，说明重组载体在C2C12中能够表达，而在CHO细胞中重组载体看不到表达，psuper则具有很高的表达。这就说明了重组载体具有骨骼肌卫星细胞表达特异性。(3)制备的逆转录病毒悬液转染C2C12后，绿色荧光表达效率有所提高但是并不非常明显。在转染了诱导分化的P19细胞中，发现了绿色荧光蛋白的表达，据此可以定性判断DMSO诱导P19细胞分化为骨骼肌细胞。 结论：通过电泳分析方法鉴定了所构建载体是正确的，另外在转染CHO和C2C12细胞的特异性验证过程中，通过绿色荧光蛋白表达结果分析说明了所构建载体具有骨骼肌卫星细胞特异性。这为骨骼肌细胞的纯化提供了基础，也为骨骼肌卫星细胞用于组织工程和基因治疗提供了基础。
[Abstract]:Aim: satellite cell (muscle satellite cells) is thought to be a myogenic cell reserved in adult skeletal muscle with proliferative and differentiation ability. To establish a simple and rapid method for the identification and screening of skeletal muscle satellite cells for satellite cell culture, Research and application are of practical significance. Desmin is now known as a marker protein expressed in skeletal muscle satellite cells and highly expressed in muscle fibers. Therefore, the reporter gene controlled by promoter element of Desmin gene can be used to identify and screen skeletal muscle satellite cells. The purpose of this study was to construct a retroviral reporter gene vector, which was specifically expressed in skeletal muscle cells, and to test the expression of green fluorescent protein in skeletal muscle cells. On this basis, the recombinant vector was prepared into retroviral suspension and its expression in differentiated P19 cells was detected. Methods: (1) Construction and identification of the recombinant vector. First, the desired target fragments, (GFP NEO) and neomycin gene (GFP NEO) and desmin promoter and enhancer (pro enh), were amplified by PCR method. Using restriction endonuclease and T4 DNA ligase, the target gene was linked to Nco 鈪
本文编号：2311745
[Abstract]:Aim: satellite cell (muscle satellite cells) is thought to be a myogenic cell reserved in adult skeletal muscle with proliferative and differentiation ability. To establish a simple and rapid method for the identification and screening of skeletal muscle satellite cells for satellite cell culture, Research and application are of practical significance. Desmin is now known as a marker protein expressed in skeletal muscle satellite cells and highly expressed in muscle fibers. Therefore, the reporter gene controlled by promoter element of Desmin gene can be used to identify and screen skeletal muscle satellite cells. The purpose of this study was to construct a retroviral reporter gene vector, which was specifically expressed in skeletal muscle cells, and to test the expression of green fluorescent protein in skeletal muscle cells. On this basis, the recombinant vector was prepared into retroviral suspension and its expression in differentiated P19 cells was detected. Methods: (1) Construction and identification of the recombinant vector. First, the desired target fragments, (GFP NEO) and neomycin gene (GFP NEO) and desmin promoter and enhancer (pro enh), were amplified by PCR method. Using restriction endonuclease and T4 DNA ligase, the target gene was linked to Nco 鈪
本文编号：2311745
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