摘要
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7 d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。
In this study,GATAIs knockout human pluripotent stem cells(hPSCs)cell line was established,and the effect of GATAIs deletion on hematopoietic differentiation in vitro was discussed.hPSCs were gene-edited to knock out GATAIs by constructing a targeting plasmid containing a recombinant arm-knock-in fragment(GATAI exonⅠ-Ⅵsequence 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 GATAIs 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 GATAIs knockout cell line.Overexpression of GATAI through transposon system also failed to rescue the reduced GPA^(+),CD41a^(+)and CD42b^(+)cells.In this study,we established GATAIs knockout hPSCs and found that GATAIs play an important role in hematopoietic differentiation.
作者
李霞
李晓红
周涯
马峰
张勇刚
LI Xia;LI Xiaohong;ZHOU Ya;MA Feng;ZHANG Yonggang(Institute of Blood Transfusion,Chinese Academy of Medical Science&Peking Union Medical College,Chengdu 610052,China)
出处
《生命的化学》
CAS
2023年第3期443-453,共11页
Chemistry of Life
基金
国家自然科学基金面上项目(82170121)
国家自然科学基金青年项目(82000119)。
