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启动子效率验证
 

启动子是基因的重要组成部分,它的主要功能是控制基因表达(转录)的起始时间和表达的程度。启动子就像“开关”,决定基因的活动。但启动子本身并不控制基 因活动,而是通过与转录因子结合而控制基因活动。启动子一般分为广谱表达型启动子、组织特异性启动子、肿瘤特异性启动子等多种形式?;虻钠舳硬糠址⑸?改变(突变),则可能导致基因表达的调节障碍。这种变化常见于恶性肿瘤。
 

  • 1.启动子是基因的重要组成部分,启动子就像“开关”,决定基因的活动;
  • 2.启动子活性的异常,则可能导致基因表达的调节障碍,从而有可能导致疾病的发生;
  • 3.找到组织特异性启动子,为靶向治疗提供可能;
  • 4.找到某些疾病关键基因异常表达与启动子的关系,为基因治疗提供可能;

 

研究背景


鉴于启动子的重要功能,目前对启动子的研究仍旧是一个热门话题。


 

启动子特点

 

  • 1.通常含一些特定的元件,如TATA box
  • 2.具有方向性:启动下游的基因的表达
  • 3.启动子长度的不确定性
  • 4.一个基因通常含有多个启动子,启动不同转录本的表达
  • 5.某些启动子具有表达的时空性和组织特异性。
  •  
 

研究思路


对启动子功能及结构的研究,可以从以下几个方面着手:

  • 1.启动子结构研究,包括核心启动子区域、正调控区域、幅调控区域及增强子的确定
  • 2.组织特异性启动子筛选及确定
  • 3.转录因子与顺式作用元件相互作用研究
  • 4.启动子甲基化作用研究
  • 5.新顺式作用元件发现及功能鉴定
  •  


研究路线



研究所涉及到的实验技术

 

  • 1.生物信息学预测;
  • 2.DNA提取及合成;
  • 3.载体构建;
  • 4.细胞培养及转染;
  • 5.双荧光检测;
  • 6.甲基化检测;
  • 7.qPCR;
  • 8.EMSA;
  • 9.chIP等。
  •  

启动子荧光素酶验证
 

pGL4启动子报告克隆在Luc2报告基因上游插入一段1.2~1.5 kb的序列,这段插入序列与基因转录起始位点(TSS)上游大约1.5kb到下游200bp之间的5'侧翼序列一致,可以通过观察荧光素酶的活性来研究启动子对基因的调节作用。

权阳生物可提供预制和定制的pGL-Promoter启动子克隆,包括单报告基因载体系统和双报告基因载体系统。单报告基因载体系统以Rluc、 mCherry或GFP作为启动子的报告基因;双报告基因载体系统以Luc作为启动子报告基因,以Rluc作为信号标准化的内参,可在活细胞中对超过15000种人和16000种小鼠启动子进行实时活性分析。




图1.pGL4 启动子报告克隆工作流程
 

双报告系统

广东快乐十分直播开奖 www.netjwr.com.cn pGL 启动子克隆可以将Rluc作为第二个报告子和内参。这种双报告系统可以对多样本常规转染进行精确比较。

 

图2. 不同启动子在H1B1B和HEK293T细胞中的活性分析。双报告系统启动子克隆和对照以两个重复转染进两种细胞中。转染24小时((HEK293T)和48小时(H1B1B)后分析样品。NEG(包含非启动子序列)和和EMPTY(载体不含启动子)作为阴性对照。

 

 

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