环状RNA mmu-circ-Pax3.1表达载体的构建及初步验证

doi:10.16431/j.cnki.1671-7236.2017.10.006

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (10): 2865-2870.doi: 10.16431/j.cnki.1671-7236.2017.10.006

环状RNA mmu-circ-Pax3.1表达载体的构建及初步验证

曹洋¹, 尤双¹, 姚洋¹, 李村院¹, 陈创夫², 倪伟¹, 胡圣伟¹

1. 石河子大学生命科学学院, 石河子 832003;
2. 石河子大学动物科技学院, 石河子 832003

收稿日期:2017-04-12 出版日期:2017-10-20 发布日期:2017-10-20
通讯作者: 胡圣伟 E-mail:hushengwei@163.com
作者简介:曹洋(1991-),男,山东枣庄人,硕士生,研究方向:动物基因工程,E-mail:534109054@qq.com
基金资助:
国家自然基金项目"绵羊胚胎骨骼肌发育相关环状RNA的筛选和功能研究"（31660644）

Construction and Preliminary Verification of Expression Vector of Circular RNA mmu-circ-Pax3.1

CAO Yang¹, YOU Shuang¹, YAO Yang¹, LI Cun-yuan¹, CHEN Chuang-fu², NI Wei¹, HU Sheng-wei¹

1. College of Life Science, Shihezi University, Shihezi 832003, China;
2. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China

Received:2017-04-12 Online:2017-10-20 Published:2017-10-20

摘要/Abstract

摘要：

试验旨在探讨利用环状RNA表达载体在体外表达环状RNA mmu-circ-Pax3.1的可行性。利用反向PCR及测序证实mmu-circ-Pax3.1的存在，将其对应的线性序列克隆到环状RNA表达载体pcDNA3.1（+） CircRNA Mini Vector中，构建重组载体pcDNA3.1（+） CircRNA-mmu-Pax3.1，经PCR、酶切、测序等对重组质粒进行鉴定。利用脂质体2000转染试剂将重组质粒转染到293细胞中，在倒置荧光显微镜下观察细胞转染情况。最后利用RT-PCR检测正常细胞组、空载体组及试验组中环状RNA mmu-circ-Pax3.1的表达情况。结果表明，试验成功构建了环状RNA mmu-circ-Pax3.1表达载体，可在293细胞中高效转录mmu-circ-Pax3.1。试验利用基因工程技术构建的环状RNA mmu-circ-Pax3.1表达载体，通过脂质体法转染293细胞，使其高效转录，为深入研究mmu-circ-Pax3.1的功能奠定了基础。

关键词: 环状RNA; 载体构建; 293细胞; 转染

Abstract:

This study was aimed to investigate the feasibility of expressing mmu-circ-Pax3.1 by circular RNA expressed vector in vitro. The exists of mmu-circ-Pax3.1 was confirmed by reverse PCR and sequenced, and constructed pcDNA3.1(+) CircRNA-mmu-Pax3.1 through cloning the corresponding linear sequence into pcDNA3.1(+) CircRNA Mini Vector. The recombinant vector was identified by PCR, enzyme digestion and sequencing methods.The recombinant plasmid was transfected into 293 cells by Lipofectamine 2000 transfection reagent, transfected cells were observed under the inverted fluorescence microscope. Finally,the expression of mmu-circ-Pax3.1 was detected by RT-PCR in normal cells group, empty vector group and experimental group. The mmu-circ-Pax3.1 expression vector was successfully constructed, and the vector could make mmu-circ-Pax3.1 efficient transcription into 293 cells. The circular RNA expression vector constructed in this experiment by genetic engineering technology was transfected into 293 cells by liposome method, and transcribed mmu-circ-Pax3.1 efficiently and laid a foundation for further study for the function of mmu-circ-Pax3.1.

Key words: circular RNA; vector construction; 293 cells; transfection

中图分类号:

曹洋, 尤双, 姚洋, 李村院, 陈创夫, 倪伟, 胡圣伟. 环状RNA mmu-circ-Pax3.1表达载体的构建及初步验证[J]. 《中国畜牧兽医》, 2017, 44(10): 2865-2870.

CAO Yang, YOU Shuang, YAO Yang, LI Cun-yuan, CHEN Chuang-fu, NI Wei, HU Sheng-wei. Construction and Preliminary Verification of Expression Vector of Circular RNA mmu-circ-Pax3.1[J]. , 2017, 44(10): 2865-2870.

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环状RNA mmu-circ-Pax3.1表达载体的构建及初步验证

Construction and Preliminary Verification of Expression Vector of Circular RNA mmu-circ-Pax3.1

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