针对ALK4基因的TALEN质粒构建与活性鉴定

生物技术通报 ›› 2014, Vol. 0 ›› Issue (5): 210-216.

• 研究报告 • 上一篇

针对ALK4基因的TALEN质粒构建与活性鉴定

曾凡才^1，2 顾洪² 王轲² 周红¹

(1.电子科技大学生命科学与技术学院，成都 610054；2.泸州医学院生物化学与分子生物学实验室，泸州 646000)

收稿日期:2013-10-31 出版日期:2014-05-23 发布日期:2014-05-24
作者简介:曾凡才，男，博士，研究方向：肿瘤分子生物学；E-mail：zfcai@sina.com
基金资助:
国家自然科学基金资助项目(30972280)，四川省教育厅自然科学基金资助项目(11ZA241)

Construction and Activity Assay of Transcription Activator-like Effector Nuclease(TALEN)Plasmids for ALK4 Gene Knock-out

Zeng Fancai^1，2 Gu Hong² Wang Ke² Zhou Hong¹

(1. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054；2. Laboratory of Biochemistry and Molecular Biology，Luzhou Medical College，Luzhou 646000)

Received:2013-10-31 Published:2014-05-23 Online:2014-05-24

摘要/Abstract

摘要： 旨在利用类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease，TALEN)技术构建具有活性的敲除类激活素激酶受体4(Activin receptor-like kinase 4，ALK4)的TALEN质粒。利用TALEN在线设计工具，根据TALEN设计原则和ALK4剪接异构体的共同序列确定基因敲除的靶位点、TALEN识别序列和用于活性验证的限制性酶切位点。利用质粒文库试剂盒快速构建TALEN质粒，并通过酶切、测序和BLAST比对加以验证。应用脂质体转染法将构建质粒导入HEK293T细胞，通过共转染的pEGFP-N1质粒判断转染效率。利用嘌呤霉素进行阳性筛选后提取基因组DNA，PCR扩增靶序列，HhaⅠ酶切纯化后的PCR产物。结果显示，来自转染TALEN质粒细胞基因组的PCR产物的酶切效率明显下降，提示部分细胞的ALK4基因发生了突变。首次成功构建了在HEK293T细胞中有活性的TALEN质粒。

关键词: 类激活素激酶受体4, 类转录激活因子效应物核酸酶, 基因敲除, HEK293T细胞, 质粒构建

Abstract: The plasmids of transcription activator-like effector nuclease(TALEN)to knock out Activin receptor-like kinase 4(ALK4)was obtained. Firstly, using a TALEN design tool online, the target sites of gene knock-out, TALEN recognition sequence and the restriction site for evaluating TALEN activity were defined according to the design rule and the common sequence of ALK4 variants. Secondly, the TALEN plasmids were constructed using a TALEN construction kit, and then confirmed by enzyme cutting, sequencing and BLAST analysis. Thirdly, the plasmids were transfected into HEK293T cells by lipofection and the transfection efficiency was assessed by observing the expression of pEGFP-N1 plasmid. After positive screening by puromycin, the genomic DNA of cells was extracted as the template, and the PCR products containing target sequence were digested by Hha I. The results showed that the cleavage efficiency of this enzyme for PCR products from genomic DNA of cells tranfected with TALEN plasmids was significantly lower than that from the cells without TALEN, suggesting that ALK4 genes may undergo mutation due to TALEN activity. These TAELN plasmids provide a valuable basis for constructing various cell lines with ALK4 knock-out and understanding the function role of ALK4.

Key words: Activin, Receptor-Like kinase 4, Transcription activator-like effector nuclease, Gene knock-out, HEK293T Cell, Plasmid Construction

曾凡才, 顾洪, 王轲, 周红. 针对ALK4基因的TALEN质粒构建与活性鉴定[J]. 生物技术通报, 2014, 0(5): 210-216.

Zeng Fancai, Gu Hong, Wang Ke, Zhou Hong. Construction and Activity Assay of Transcription Activator-like Effector Nuclease(TALEN)Plasmids for ALK4 Gene Knock-out[J]. Biotechnology Bulletin, 2014, 0(5): 210-216.

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针对ALK4基因的TALEN质粒构建与活性鉴定

Construction and Activity Assay of Transcription Activator-like Effector Nuclease(TALEN)Plasmids for ALK4 Gene Knock-out

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