小鼠中枢神经系统载脂蛋白C2启动子的克隆及转录

doi:10.3969/j.issn.1673-5374.2013.02.008

摘要/Abstract

摘要：

实验对小鼠参与中枢神经系统中脂类的转运和代谢的载脂蛋白C2启动子区域进行转录活性分析，构建了载脂蛋白C2转录起始点约2kb范围内含7种不同DNA片段的萤光素酶表达质粒，7种萤光素酶表达质粒分别转染293T细胞，载脂蛋白C2各区段启动子转录活性检测，通过生物信息学数据库分析小鼠载脂蛋白C2启动子区域的转录因子结合位点，确定了载脂蛋白C2核心启动子区域为+104bp～+470bp区域。+104bp～+45bp含有较强的正性调节元件，-894bp～-497bp含有较弱的正性调节元件，而在-1350bp～-894bp和-497bp～-214bp含有较强的负性调节元件，-214bp～+45bp区域含有较弱的负性调节元件，这种正负调控区交错的启动子活性现象表明了小鼠载脂蛋白C2基因表达调控的复杂性。

关键词: 神经再生, 基础实验, 载脂蛋白C2, 启动子, 双荧光素酶报告基因检测, 转录活性, 调节元件, 脑, 神经系统, 小鼠, 再生, 基金资助文章

Abstract:

Apolipoprotein C2 is an important member of the apolipoprotein C family, and is a potent activator of lipoprotein lipase. In the central nervous system, apolipoprotein C2 plays an important role in the catabolism of triglyceride-rich lipoproteins. Studies into the exact regulatory mechanism of mouse apolipoprotein C2 expression have not been reported. In this study, seven luciferase expression vectors, which contained potential mouse apolipoprotein C2 gene promoters, were constructed and co-transfected with pRL-TK into HEK293T cells to investigate apolipoprotein C2 promoter activity. Luciferase assays indicated that the apolipoprotein C2 promoter region was mainly located in the +104 bp to +470 bp region. The activity of the different lengths of apolipoprotein C2 promoter region varied. This staggered negative-positive-negative arrangement indicates the complex regulation of apolipoprotein C2 expression and provides important clues for elucidating the regulatory mechanism of apolipoprotein C2 gene transcription.

Key words: neural regeneration, basic research, apolipoprotein C2, promoter, dual-luciferase reporter assay, transcriptional activity, regulatory elements, grants-supported paper, neuroregeneration

. 小鼠中枢神经系统载脂蛋白C2启动子的克隆及转录[J]. 中国神经再生研究(英文版), 2013, 8(2): 156-161.

Zhaoyang Li, Bing Du, Shengyang Li, Xiangchuan Lv, Shenglai Zhou, Yang Yu, Wei Wang, Zhihong Zheng. Cloning and characterization of an apolipoprotein C2 promoter in the mouse central nervous system[J]. Neural Regeneration Research, 2013, 8(2): 156-161.

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引言

材料方法

Design

An engineered cytological controlled experiment.

Time and setting

Experiments were performed at the Department of Laboratory Animals of China Medical University, China from 2009 to 2011.

Materials

Human embryonic kidney cells (HEK293T) and C57BL/6J mice were supplied by the Department of Laboratory Animals of China Medical University, China.

Methods

Cell culture

HEK293T were grown in RPMI-1640 containing 10% heat inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), at 37°C in 5% CO₂. When the cells were in the logarithmic phase, they were cultured on 96-well plates. All cell culture reagents other than fetal bovine serum were purchased from Invitrogen, Carlsbad, CA, USA.

Bioinformatics analysis and PCR primer design

We analyzed the region of apolipoprotein C2 (GenBank Gene ID: 11813) –2 000 bp to +700 bp using http:// www.fruitfly.org/seq_tools/promoter.html, and designed seven pairs of PCR primers based on the results:

Construction of mouse apolipoprotein C2 promoter-luciferase reporter

C57BL/6J mouse tail genomic DNA was used as a PCR template, and seven promoter segments with different lengths were amplified using PCR. Plasmid pGL3-basic and the seven promoter DNAs were double digested with restriction enzymes KpnI and XhoI. Several colonies were obtained after ligation and transformation. The colonies were identified by PCR, restriction enzyme and sequence analysis. The successfully constructed plasmids were named pGL3-Basic-apolipoprotein C2-A (2 430 bp), pGL3-Basic-apolipoprotein C2-B (1 821 bp), pGL3-Basic-apolipoprotein C2-C (1 365 bp), pGL3-Basic-apolipoprotein C2-D (968 bp), pGL3-Basic-apolipoprotein C2-E (685 bp), pGL3-Basic-apolipoprotein C2-F (425 bp), and pGL3-Basic-apolipoprotein C2-G (367 bp).

Transient transfection

pGL3-Basic-apolipoprotein C2-X and pRL-TK were co-transfected into 293T cells under the mediation of Lipofectamine^TM2000 (Invitrogen). pGL3-Basic- apolipoprotein C2-X plasmid was 200 ng/well, and pRL-TK was 20 ng/well, and 10% fetal bovine serum was added 4 hours after co-transfection.

Luciferase assays

Transfected cells were incubated for 48 hours at 37°C and washed three times with PBS. Cells were lysed and the dual-luciferase assay was performed according to the manufacturer’s instructions (Promega, Madison, WI, USA). Each transfection was performed in triplicate and experiments were carried out a total of three times.

Transcriptional factor binding sites of the apolipoprotein C2 promoter

Transcription factor binding sites of the apolipoprotein C2 promoter –1 350 bp to +470 bp were analyzed through http://www.cbil.upenn.edu/cgi-bin/tess/tess.

Statistical analysis

All experimental data were analyzed using the SPSS 17.0 software (SPSS, Chicago, IL, USA). The differences in the mean value between groups were compared using one-way analysis of variance. Paired comparisons of the mean value between groups were performed using a two-sample t-test. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1实验设计及文章构思：
首先进行C57BL/6J小鼠鼠尾基因组DNA的提取，之所以选择C57BL/6J小鼠，是因为此品系小鼠为近交系小鼠，已经获得其全基因组序列，遗传背景明确，为以后通过动物水平研究载脂蛋白C2基因功能奠定基础。

载脂蛋白C2启动子区域的克隆及萤光素酶表达质粒的构建，以C57BL/6J小鼠鼠尾基因组DNA为模板，分别扩增不同长度的7段启动子区域的DNA片段，通过分子生物学方法构建萤光素酶表达质粒。

启动子不同区域转录活性分析，含载脂蛋白C2基因不同启动子DNA 片段的萤光素酶报告基因质粒pGL3-ApoC2-X和内参照pRL-TK共质粒转染293T细胞，进行萤光素酶活性分析。

载脂蛋白C2基因启动子区域转录因子结合位点分析，使用http://www.cbil.upenn.edu/cgi-bin/tess/tess数据库对载脂蛋白C2基因启动子区域进行转录因子结合位点分析。

2文章学术水平与国内外同类研究的比较：
研究使用荧光素酶报告基因系统对载脂蛋白C2基因启动子转录活性进行分析，此方法是目前国际上此类研究的主流方法，本研究首次分析了小鼠载脂蛋白C2基因上游序列不同区域的转录活性及转录因子结合位点，确定了载脂蛋白C2基因核心启动子区域，初步揭示了载脂蛋白C2基因上游序列的结构及功能特征。

3实验设计构思简介：
载脂蛋白C2是载脂蛋白家族中最重要的亚型之一。在哺乳动物神经系统中，载脂蛋白C2与脂类转运和代谢相关。载脂蛋白C2异常还与阿尔茨海默病、脂性脑病和12型脊髓小脑共济失调有关。目前对小鼠载脂蛋白C2基因自身的转录调控未见报道，分析小鼠载脂蛋白C2基因启动子区域结构、功能特征对于通过小鼠动物模型研究载脂蛋白C2基因在神经系统中的表达调控具有重要的意义。

实验克隆小鼠载脂蛋白C2基因启动子区域不同长度的DNA片段进行转录活性检测，通过生物信息学数据库对小鼠载脂蛋白C2基因启动子区域的转录因子结合位点进行分析，初步分析了小鼠载脂蛋白C2基因上游序列不同区域转录活性的差异，确定了载脂蛋白C2基因核心启动子区域，初步揭示了载脂蛋白C2基因上游序列的结构及功能特征，为进一步研究载脂蛋白C2的表达调控奠定基础。

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