首页|GATA1s敲除hPSCs模型构建与造血分化影响初探

GATA1s敲除hPSCs模型构建与造血分化影响初探

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本文建立了GATA1s敲除人多能干细胞(human pluripotent stem cells,hPSCs)细胞株,并以此为模型探讨了GATA1s缺失对体外诱导造血分化的影响.本文通过构建含有重组臂-敲入片段(GATA1外显子Ⅱ-Ⅵ序列及BGH polyA序列)-LoxP-潮霉素筛选标记-LoxP-重组臂的打靶质粒和含有gRNA-Cas9的gRNA质粒对hPSCs进行基因编辑以敲除GATA1s.通过第一步电穿孔将以上质粒导入细胞,潮霉素初步筛选7d后的阳性克隆进行下一步电穿孔环化重组酶(Cre)使LoxP位点特异性重组以去除筛选标记,通过单细胞克隆的方式进一步培养,PCR及测序鉴定出正确基因编辑细胞,最后通过蛋白质免疫印迹验证其GATA1与GATA1s蛋白的表达情况,验证敲除GATA1s后的细胞株进行体外诱导造血分化,发现其CD34+、CD43+及CD45+细胞增多,但GPA+、CD41a+及CD42b+细胞显著减少,通过转座子系统过表达GATA1也不能挽救其减少的GPA+、CD41a+及CD42b+细胞.本研究建立了GATA1s敲除hPSCs细胞株,并发现其在造血分化时有重要作用.
Construction of GATA1s knockout hPSCs model and its effect on hematopoietic differentiation
In this study,GATA1s knockout human pluripotent stem cells(hPSCs)cell line was established,and the effect of GATA1s deletion on hematopoietic differentiation in vitro was discussed.hPSCs were gene-edited to knock out GATA1s by constructing a targeting plasmid containing a recombinant arm-knock-in fragment(GATA1 exon II-VI sequence and BGH polyA sequence)-LoxP-hygromycin screening marker-LoxP-recombination arm and a gRNA plasmid containing gRNA-Cas9.The above plasmids were introduced into the cells by electroporation.After 7 days of initial screening with hygromycin,the positive clones were subjected to electroporation to make the LoxP site specificially recombined by Cre to remove the screening marker,and further cultured by single-cell cloning.Finally,the expression of GATA1 and GATA1s proteins was verified by Western blot,and the CD34+,CD43+ and CD45+ cells were increased,but GPA+,CD41a+ and CD42b+ cells were significantly decreased in the GATA1s knockout cell line.Overexpression of GATA1 through transposon system also failed to rescue the reduced GPA+,CD41a+and CD42b+cells.In this study,we established GATA1s knockout hPSCs and found that GATA1s play an important role in hematopoietic differentiation.

GATA1GATA1smultipotent stem cellsgene knockin techniquesclustered regularly interspaced short palindromic repeatsCRISPR associated proteinshematopoietic differentiation

李霞、李晓红、周涯、马峰、张勇刚

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中国医学科学院北京协和医学院输血研究所,成都 610052

GATA1 GATA1s 多能干细胞 基因敲入技术 聚簇规则间隔短回文重复序列 CRISPR相关蛋白 造血分化

国家自然科学基金面上项目国家自然科学基金青年项目

8217012182000119

2023

10.13488/j.smhx.20230212

生命的化学
中国生物化学与分子生物学会

生命的化学

CSTPCD
影响因子：0.404
ISSN：1000-1336
年,卷(期)：2023.(3)
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