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

Abstract:

OBJECTIVES: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. RESULTS: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. CONCLUSIONS: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.

journal_name

Biotechnol Lett

journal_title

Biotechnology letters

authors

Chang Y,Shao J,Gao Y,Liu W,Gao Z,Hu Y,Chang H

doi

10.1007/s10529-020-02860-x

subject

Has Abstract

pub_date

2020-08-01 00:00:00

pages

1317-1325

issue

8

eissn

0141-5492

issn

1573-6776

pii

10.1007/s10529-020-02860-x

journal_volume

42

pub_type

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