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

Abstract

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

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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