Abstract: Runt-related transcription factor 1c (Runx1c) is closely related to definitive hematopoiesis and the production of hematopoietic stem cell (HSC). In order to track the dynamical expression of Runx1c during early hematopoietic development, Runx1c-mNeonGreen reporter cell line derived from human embryonic stem cell (hESC) was efficiently constructed by CRISPR-Cas9 technology combined double-cut plasmid vector with the transient transfer of B-cell lymphoma-extra large (BCL-XL) gene. It was identified and verified by PCR and Sanger sequencing, and the results showed that the construction was successful. Subsequently, immunofluorescence technique, real-time fluorescence quantitative PCR technique, flow cytometry, teratoma assay and digital karyotype analysis technique were used to verify that the reporter cell line still had hESC-like pluripotency and no chromosome abnormality or variation occurred. In addition, the expression of Runx1c in human early hematopoietic development was tracked by flow cytometry through hESC in vitro monolayer induced hematopoietic differentiation. The results showed that the reporter cell line could dynamically track the expression of Runx1c in human early hematopoietic development. The research results provided a new idea for elucidating definitive hematopoiesis and the possibility of producing functional blood cells in vitro.

Key words: human embryonic stem cells, definitive hematopoiesis, Runx1c, reporter cell line, CRISPR-Cas9

摘要： Runt相关转录因子1c（Runt-related transcription factor 1c，Runx1c）与成年造血以及造血干细胞（hematopoietic stem cell，HSC）的产生密切相关，为了动态追踪人早期造血发育过程中Runx1c的表达，利用双切质粒载体联合瞬时转入B细胞淋巴瘤-特大（B-cell lymphoma-extra large，BCL-XL）基因的CRISPR-Cas9技术高效地构建了人胚胎干细胞（human embryonic stem cell，hESC）来源的Runx1c-mNeonGreen报告细胞系，并经PCR、Sanger测序验证构建成功。随后，利用免疫荧光技术、实时荧光定量PCR技术、流式细胞术、畸胎瘤实验和数字化核型分析技术进行验证，发现该报告细胞系仍具有hESC样的多能性且未发生染色体异常和变异。此外，通过hESC体外单层诱导造血分化方法，利用流式细胞术追踪人早期造血发育过程中Runx1c的表达，结果表明利用该报告细胞系可以动态追踪人早期造血发育中Runx1c的表达。研究结果为阐明成年造血过程提供了新思路，并为体外产生功能性血细胞提供了可能。

关键词: 人胚胎干细胞, 成年造血, Runx1c, 报告细胞系, CRISPR-Cas9