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内部核糖体进入位点(IRES) — 实现同一载体高效共表达两个基因的元件

简介

 IRES是实现同一载体高效共表达两个基因的元件。一般可以通过装载荧光标签或可以在表达的同时生物素化的蛋白到同一载体来检测一个基因的转染效率。IRES元件可以用于以下研究:

  • 检测基因转染效率 
  • 检测蛋白修饰 
  • 体内生物素化 
  • 稳定转染 

 

OmicsLink ORF cDNA clones with IRES element

 

技术

IRES技术可以使用同一启动子在同一载体高效共表达两个基因的元件 (见figures 1和 2)。GeneCopoeia已经构建了许多包含IRES元件的载体。

 

Combined with OmicsLink expression-ready clones

为了满足不同需求,GeneCopoeia已经将IRES元件构建到 部分哺乳动物细胞和慢病毒载体,有以下报告基因可选:

  • eGFP
  • mCherry
  • SUMO
  • 生物素连接酶
  • 新霉素
  • 荧光素酶

 

 GeneCopeia提供带有IRES元件、CMV 启动子的20,000多种人和15,000多种小鼠的表达克隆。

Choice of co-expressing any reporter gene with IRES element

 

优势

  • 可以将报告基因对靶蛋白的生物学活性的影响降到最低
  • 可以有效共转染因为两个基因在同一载体

 

 

 

 

 

OmicsLink ORF cDNA clones with IRES element

 

 

 

 

图1. 带有IRES元件的OmicsLink™ 即用型ORF表达表达克隆

应用实例

应用

  • 使报告基因如 eGFP、mCherry 或 Luciferase与靶蛋白共表达以检测转染效率
  • 可以通过共表达如SUMO这样特定的蛋白修饰酶来监测蛋白修饰
  • 共表达生物素连接酶可以在体内生物素化带有AviTag的蛋白
  • 共表达如Neomycin这样的筛选标记可以进行稳定细胞株的筛选

 

严格的质检和质量保证

OmicsLink clone collection里的所有ORF表达克隆源于验证过的全长cDNA克隆或高质量的人源cDNA组织文库。

寄送之前,GeneCopoeia对所有的克隆进行了严格的质检过程。

  • 所有的ORF经过全长测序
  • 通过PCR验证序列长度
  • 通过酶切验证质粒的正确性

IRES作用流程

Schematic of protein expression with IRES technology

Specific biotinylation of AviTag eGFP by E. coli biotin ligase in 293 cells

图2. 在293细胞通过大肠杆菌生物素连接酶特异性的生物素化AviTag

载体信息

使用IRES技术的表达载体列表

载体 启动子 宿主细胞 筛选标记 标签 蛋白酶位点
pReceiver-Lv165 EF1a Stem/primary/others* N/A IRES2-eGFP N/A
pReceiver-Lv166 EF1a Stem/primary/others* N/A IRES2-mcherry N/A
pReceiver-Lv201 CMV Stem/primary/others* Puromycin SV40-eGFP-IRES-puromycin N/A
pReceiver-Lv202 CMV Stem/primary/others* Puromycin C-Myc-SV40-eGFP-IRES-puromycin N/A
pReceiver-Lv203 CMV Stem/primary/others* Puromycin C-Flag-SV40-eGFP-IRES-puromyci N/A
pReceiver-Lv204 CMV Stem/primary/others* Puromycin C-Myc-SV40-mCherry-IRES-puromy N/A
pReceiver-Lv205 CMV Stem/primary/others* Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-Lv206 CMV Stem/primary/others* Puromycin SV40-mCherry-IRES-puromycin N/A
pReceiver-Lv207 CMV Stem/primary/others* Hygromycin SV40-eGFP-IRES-hygromycin N/A
pReceiver-Lv211 CMV Stem/primary/others* Hygromycin C-Halotag-IRES-hygromycin N/A
pReceiver-Lv213 CMV Stem/primary/others* Puromycin IRES2-mCherry-IRES-puromycin N/A
pReceiver-Lv214 CMV Stem/primary/others* N/A IRES2-mCherry N/A
pReceiver-Lv215 CMV Stem/primary/others* N/A IRES2-eGFP N/A
pReceiver-Lv216 CMV Stem/primary/others* Puromycin C-Flag-SV40-mCherry-IRES-purom N/A
pReceiver-Lv217 CMV Stem/primary/others* Puromycin SV40-hLUC-IRES-puromycin N/A
pReceiver-Lv220 CMV Stem/primary/others* Hygromycin IRES2-Hygromycin N/A
pReceiver-Lv224 EF1a Stem/primary/others* Puromycin IRES2-mcherry-IRES-puromycin N/A
pReceiver-Lv225 EF1a Stem/primary/others* Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-Lv228 PGK Stem/primary/others* N/A IRES2-eGFP N/A
pReceiver-Lv229 PGK Stem/primary/others* N/A IRES2-mcherry N/A
pReceiver-Lv230 PGK Stem/primary/others* Puromycin IRES2-mcherry-IRES-puromycin N/A
pReceiver-Lv231 PGK Stem/primary/others* Puromycin IRES2-eGFP-IRES-puromycin N/A
pReceiver-M45 CMV Mammalian Neomycin C-3xHA+IRES-eGFP N/A
pReceiver-M46 CMV Mammalian Neomycin C-Flag+IRES-eGFP N/A
pReceiver-M48 CMV Mammalian Neomycin N-Avi+IRES-Biotin ligase N/A
pReceiver-M51 CMV Mammalian Neomycin C-His+IRES-eGFP N/A
pReceiver-M61 CMV Mammalian Neomycin IRES2-eGFP N/A
pReceiver-M72 CMV Mammalian Neomycin C-Myc+IRES2-eGFP N/A

 

点击了解即用型ORF克隆 ,提供大肠杆菌、哺乳动物细胞、昆虫细胞、酵母和麦胚无细胞表达克隆载体和50种融合标签。

 

 

参考文献

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