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啟動子效率驗證
 

啟動子是基因的重要組成部分,它的主要功能是控制基因表達(轉錄)的起始時間和表達的程度。啟動子就像“開關”,決定基因的活動。但啟動子本身并不控制基 因活動,而是通過與轉錄因子結合而控制基因活動。啟動子一般分為廣譜表達型啟動子、組織特異性啟動子、腫瘤特異性啟動子等多種形式。基因的啟動子部分發生 改變(突變),則可能導致基因表達的調節障礙。這種變化常見于惡性腫瘤。
 

  • 1.啟動子是基因的重要組成部分,啟動子就像“開關”,決定基因的活動;
  • 2.啟動子活性的異常,則可能導致基因表達的調節障礙,從而有可能導致疾病的發生;
  • 3.找到組織特異性啟動子,為靶向治療提供可能;
  • 4.找到某些疾病關鍵基因異常表達與啟動子的關系,為基因治療提供可能;

 

研究背景


鑒于啟動子的重要功能,目前對啟動子的研究仍舊是一個熱門話題。


 

啟動子特點

 

  • 1.通常含一些特定的元件,如TATA box
  • 2.具有方向性:啟動下游的基因的表達
  • 3.啟動子長度的不確定性
  • 4.一個基因通常含有多個啟動子,啟動不同轉錄本的表達
  • 5.某些啟動子具有表達的時空性和組織特異性。
  •  
 

研究思路


對啟動子功能及結構的研究,可以從以下幾個方面著手:

  • 1.啟動子結構研究,包括核心啟動子區域、正調控區域、幅調控區域及增強子的確定
  • 2.組織特異性啟動子篩選及確定
  • 3.轉錄因子與順式作用元件相互作用研究
  • 4.啟動子甲基化作用研究
  • 5.新順式作用元件發現及功能鑒定
  •  


研究路線



研究所涉及到的實驗技術

 

  • 1.生物信息學預測;
  • 2.DNA提取及合成;
  • 3.載體構建;
  • 4.細胞培養及轉染;
  • 5.雙熒光檢測;
  • 6.甲基化檢測;
  • 7.qPCR;
  • 8.EMSA;
  • 9.chIP等。
  •  

啟動子熒光素酶驗證
 

pGL4啟動子報告克隆在Luc2報告基因上游插入一段1.2~1.5 kb的序列,這段插入序列與基因轉錄起始位點(TSS)上游大約1.5kb到下游200bp之間的5'側翼序列一致,可以通過觀察熒光素酶的活性來研究啟動子對基因的調節作用。

權陽生物可提供預制和定制的pGL-Promoter啟動子克隆,包括單報告基因載體系統和雙報告基因載體系統。單報告基因載體系統以Rluc、 mCherry或GFP作為啟動子的報告基因;雙報告基因載體系統以Luc作為啟動子報告基因,以Rluc作為信號標準化的內參,可在活細胞中對超過15000種人和16000種小鼠啟動子進行實時活性分析。




圖1.pGL4 啟動子報告克隆工作流程
 

雙報告系統

pGL 啟動子克隆可以將Rluc作為第二個報告子和內參。這種雙報告系統可以對多樣本常規轉染進行精確比較。

 

圖2. 不同啟動子在H1B1B和HEK293T細胞中的活性分析。雙報告系統啟動子克隆和對照以兩個重復轉染進兩種細胞中。轉染24小時((HEK293T)和48小時(H1B1B)后分析樣品。NEG(包含非啟動子序列)和和EMPTY(載體不含啟動子)作為陰性對照。

 

 

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