Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination.

Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells. Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression. Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.

Citation

Yanyan Chang, Junjun Shao, Yuan Gao, Wei Liu, Zhan Gao, Yonghao Hu, Huiyun Chang. Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination. Biotechnology letters. 2020 Aug;42(8):1317-1325

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PMID: 32185620

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