神经细胞特异性Cre重组酶表达载体的构建及其体外表达研究

发布时间：2018-05-16 11:32

本文选题：条件性基因敲除 + 载体构建　；参考：《福建医科大学》2008年硕士论文

【摘要】： Cre-loxP系统介导的条件性基因敲除(conditional gene knockout)是基因功能研究最重要的技术之一,它克服了传统基因敲除导致小鼠胚胎死亡等诸多弊端。将该技术应用于神经系统,可使基因敲除仅发生于大脑的某个亚区或发育过程的某个阶段,以便对小鼠大脑基因功能进行精细研究。对于神经系统条件性基因敲除而言,选择一个大脑亚区和时间特异性启动子指导Cre的表达是至关重要的。许多研究表明,CaMKⅡα基因的表达具有较强的时间和空间特异性[1-4],因此,符合制备前脑神经细胞特异性表达Cre转基因小鼠的要求。本研究通过克隆和鉴定CaMKⅡα基因5’端不同调节区的序列,构建了两个神经细胞特异性表达Cre的真核表达载体,并对这两个载体的转录活性进行了初步研究。为建立前脑神经细胞特异性表达Cre的转基因小鼠及进一步制备ADAM10基因条件性敲除的小鼠奠定了基础,有助于开展阿尔茨海默病发病机制的研究。运用PCR技术,从C57BL/6J小鼠基因组中分步扩增了CaMKⅡα基因5’端两段调控序列,长度分别为5.26kb和8.1kb。经部分测序鉴定后,将片段分别连接于pCre-EGFP载体的Cre基因上游,构建了神经细胞特异性表达Cre重组酶的表达载体pCaMKⅡα-Cre-EGFPⅠ(pCCEⅠ)和pCaMKⅡα-Cre-EGFPⅡ(pCCEⅡ)。取C57BL/6J新生鼠与4周龄小鼠的端脑与海马进行神经元的原代培养。分别以pCCEⅠ和pCCEⅡ为实验组,pCre-EGFP为阳性对照组,采用脂质体2000(LP2000)转染神经细胞、小鼠神经瘤与大鼠神经胶质瘤之融合细胞(NG108-15)和人乳腺癌细胞(ZR-75-30),研究pCCEⅠ和pCCEⅡ的表达活性及其组织特异性。结果显示,克隆的两个CaMKⅡα基因5’端侧翼区片段的酶切图谱与公布的C57BL/6J小鼠相应序列的酶切位点相一致,测序结果表明,所测序列与数据库公布的C57BL/6J小鼠相应序列基本相同,仅在-940bp(A→C)和-1.4kb(A→G)两处存在差异。其中,文献报道的富含顺式调控元件的-900bp内序列[5]与公布的C57BL/6J小鼠序列完全一致。5.26kb与8.1kb这两段CaMKⅡα基因启动子均正确地连接于Cre基因的5’端,成功构建了目标载体。新生鼠神经细胞的原代培养表明,细胞数量多(35mm培养皿中细胞密度"g106/cm3),细胞形态好。4周龄鼠神经细胞的原代培养细胞形态较典型,但细胞数量很少(35mm培养皿中细胞不超过300个)。用LP2000转染后,在NG108-15细胞中,pCCEⅠ组能表达绿色荧光(表达率低),pCCEⅡ组不表达绿色荧光,阳性对照组表达绿色荧光;在ZR-75-30细胞中,pCCEⅠ组和pCCEⅡ组都不表达绿色荧光,阳性对照组表达绿色荧光;在新生鼠神经细胞中,pCCEⅠ组和pCCEⅡ组都不表达绿色荧光,阳性对照组表达绿色荧光(转染效率低);在4周龄鼠神经细胞中,由于细胞数量少,培养困难,经LP2000处理后即发生死亡,故实验组与阳性对照组均未表达绿色荧光。综上所述,得出以下结论: 本研究成功构建并鉴定了神经细胞特异性Cre表达载体(pCCEⅠ和pCCEⅡ)。成功培养了新生鼠与4周龄鼠的皮质及海马神经细胞。pCCEⅠ能在神经细胞瘤-神经胶质瘤细胞NG108-15中表达绿色荧光,说明pCCEⅠ的启动子CaMKIIα基因5.26kb片段具有启动转录的活性;pCCEⅠ和pCCEⅡ在人乳腺癌细胞ZR-75-30中不表达,支持了其表达的神经细胞特异性;pCCEⅠ和pCCEⅡ在新生鼠神经细胞中不表达,一定程度上支持了其表达的时间特异性。pCCEⅡ中包含的CaMKIIα基因几乎所有调控区的8.1kb片段的转录活性尚有待体内实验的证实。实验结果证明本研究所构建的神经细胞特异性表达Cre的转基因载体pCCEⅠ具有转录活性,且支持其表达的时空特异性。理论上证明了建立前脑神经细胞特异性表达Cre重组酶的转基因小鼠的可行性。
[Abstract]:The Cre-loxP system mediated conditional gene knockout is one of the most important techniques for gene function research. It overcomes the disadvantages of the traditional gene knockout, which leads to the death of the mouse embryo, and the application of this technique to the nervous system can only be used in some subregion of the brain or a certain stage of development. In order to make a fine study of gene function in the brain of mice, it is essential to select a subregion of the brain and a time specific promoter to guide the expression of Cre for the conditional gene knockout of the nervous system. Many studies have shown that the expression of the CaMK II alpha gene has a strong time and spatial specific [1-4], thus conforming to the preparation of the gene. Forebrain neurons specifically expressed Cre transgenic mice.
In this study, by cloning and identifying the sequence of the different regulation areas of the 5 'end of the CaMK II - a gene, two eukaryotic expression vectors expressing the specific expression of Cre were constructed, and the transcriptional activity of these two vectors was preliminarily studied. In order to establish the transgenic mice with the specific expression of Cre in the forebrain nerve cells and to further prepare the ADAM10 gene strip Knockout mice laid the foundation for the study of the pathogenesis of Alzheimer's disease.
PCR technique was used to amplify the two segments of the 5 'end of the CaMK II alpha gene from the C57BL/6J mouse genome, and the length was 5.26kb and 8.1kb. were identified by partial sequencing. The fragments were connected to the Cre gene of the pCre-EGFP vector respectively, and the expression vector of the Cre recombinant enzyme was constructed, and the expression vector of pCaMK II alpha -Cre-EGFP I was constructed. (pCCE I) and pCaMK II alpha -Cre-EGFP II (pCCE II). Primary culture of neurons in the end brain and hippocampus of 4 weeks old mice and 4 weeks old mice was cultured. PCCE I and pCCE II were used as experimental group, pCre-EGFP as positive control group, liposome 2000 (LP2000) transfected nerve cells, mouse neuroma and rat glioma fusion cells (NG). 108-15) and human breast cancer cells (ZR-75-30), to study the expression activity and tissue specificity of pCCE I and pCCE II.
The results showed that the cloned two CaMK II alpha gene 5 'end flanking region fragment was consistent with the corresponding sequence of the published C57BL/6J mice. The sequencing results showed that the sequence was basically the same as the corresponding sequence of C57BL/6J mice published by the database, and there were only two differences in -940bp (A to C) and -1.4kb (A to G). The -900bp internal sequence [5] rich in cis regulation elements and the published C57BL/6J mouse sequence fully conformed to the sequence of.5.26kb and 8.1kb, the two segment CaMK II alpha gene promoter correctly connected to the 5 'of the Cre gene and successfully constructed the target carrier. The primary culture of the newborn rat neural cells showed that the number of cells was much (the cell in 35mm culture dish). Density "g106/cm3"), the cell morphology of.4 weeks old rat neural cells is more typical, but the number of cells is little (no more than 300 cells in 35mm culture dish). After LP2000 transfection, in NG108-15 cells, pCCE I group can express green fluorescence (low expression rate), pCCE II group does not express green fluorescence, and the positive control group is green. In ZR-75-30 cells, both pCCE I group and pCCE II group did not express green fluorescence, and the positive control group expressed green fluorescence. In the newborn rat neural cells, both pCCE I group and pCCE II group did not express green fluorescence, and the positive control group expressed green fluorescence (low transfection efficiency). In the 4 week old rat neural cells, the number of cells was less and the culture was poor. It is difficult to die after LP2000 treatment. Therefore, green fluorescence was not expressed in both the experimental group and the positive control group.
To sum up, the following conclusions are drawn:
This study successfully constructed and identified the neural cell specific Cre expression vector (pCCE I and pCCE II). The cortical and hippocampal neurons of the 4 week old rats were successfully cultured and.PCCE I could express the green fluorescence in the neuroglioma cell NG108-15 cell NG108-15, indicating that the CaMKII alpha gene 5.26kb fragment of the promoter of pCCE I has a starting point. The activity of transcriptional transcription; pCCE I and pCCE II are not expressed in human breast cancer cell ZR-75-30, supporting their expressed Neurocellular specificity; pCCE I and pCCE II are not expressed in neonatal rat neural cells, and to some extent support the 8.1kb fragment of the CaMKII a gene contained in the time specific.PCCE II expressed in the expression of the 8.1kb fragment of the CaMKII a gene. The transcriptional activity has yet to be confirmed in the experiment in vivo. The experimental results show that the transgenic vector pCCE I, which is specifically expressed by Cre, has the transcriptional activity and supports the spatio-temporal specificity of its expression. The feasibility of establishing transgenic mice with the specific expression of Cre recombinant enzyme in the forebrain neural cells is theoretically proved.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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