Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination

Ying Huang; Hongchun Wu; Xinglong Han; Jie Wu; Miao Yu; Zhen-Ao Zhao; Zhenya Shen; Shijun Hu; Wei Lei

Stem Cell Research (Apr 2021)

Generation of an EFNB2-2A-mCherry reporter human embryonic stem cell line using CRISPR/Cas9-mediated site-specific homologous recombination

Ying Huang,
Hongchun Wu,
Xinglong Han,
Jie Wu,
Miao Yu,
Zhen-Ao Zhao,
Zhenya Shen,
Shijun Hu,
Wei Lei

Affiliations

Ying Huang: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Hongchun Wu: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Xinglong Han: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Jie Wu: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Miao Yu: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Zhen-Ao Zhao: Institute of Microcirculation & Department of Pathophysiology of Basic Medical College, Hebei North University, Zhangjiakou 075000, China; Hebei Key Laboratory of Critical Disease Mechanism and Intervention, Zhangjiakou 075000, China
Zhenya Shen: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China
Shijun Hu: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China; Corresponding authors.
Wei Lei: Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China; Corresponding authors.

Journal volume & issue: Vol. 52
p. 102241

Abstract

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Ephrin B2 (EFNB2) is the first identified and most widely used marker for arterial endothelial cells (AECs). We generated a heterozygous EFNB2-2A-mCherry reporter H1 cell line, H1-EFNB2-2A-mCherry+/− (WAe001-A-57), by CRISPR/Cas9-mediated insertion of 2A-mCherry cassette into the EFNB2 gene locus, immediately before the translation stop codon. The H1-EFNB2-2A-mCherry reporter cells were pluripotent and could differentiate into all three germ layer lineages. Simultaneous expression of mCherry was observed when expression of EFNB2 was increased during endothelial cell differentiation. Thus, the generated reporter cells enable live identification of EFNB2-positive AECs, and screening of small molecule compound and target genes that promote AEC differentiation.

Published in Stem Cell Research

ISSN: 1873-5061 (Print); 1876-7753 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Biology (General)
Website: https://www.journals.elsevier.com/stem-cell-research

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