弥漫大B细胞淋巴瘤OCI-Ly3细胞中TALEN介导的microRNA-21基因敲除

Knockout of Micro-RNA-21 Gene in DLBCL OCI-Ly3 cells by TALEN Technique

目的:在转染效率极低的血液肿瘤细胞株中探索敲除microRNA基因的有效方法,以方便microRNA基因在血液肿瘤疾病中功能的研究。方法:应用转录激活因子样效应蛋白核酸酶(Transcription activator-like effector nuclease,TALEN)介导的基因工程技术敲除人类弥漫大B细胞淋巴瘤OCI-Ly3细胞中的microRNA-21(miR-21)基因,然后通过流式细胞仪富集e GFP+表达细胞、PCR筛选和microRNA定量检测,筛选并建立不表达miR-21的OCI-Ly3单细胞克隆。最后,对不表达miR-21的OCI-Ly3单细胞克隆进行miR-21基因测序。结果:通过两轮的电转染和筛选实验,获得了4个几乎不表达miR-21的OCI-Ly3单细胞克隆,突变率约为实验起始细胞数的十万分之一。对突变克隆的测序结果显示,多种miR-21序列缺失或插入均可能导致该基因表达显著下调。结论:本方法可以推广、应用于在转染效率极低的血液肿瘤细胞株中敲除任何感兴趣的microRNA基因。

Objective:To explore an efficient way to knockout microRNA genes in hemapoietic cell lines with a very low transfection efficiency,so as to facilitate the study of microRNA function in hematopoietic malignancies.Methods:TALE-nucleases was utilized to knockout the microRNA-21 gene in human diffuse large B-cell lymphoma cells(OCI-Ly3).The OCI-Ly3 single cell clones without expression of miR-21 were established through eGFP^+ enrichment,PCR screening,and microRNA quantification.Finally,the miR-21 changes of mutant clones were identified by sequencing.Results:Four miR-21-knockouted OCI-Ly3 single-cell-derived clones were established after 2 round transfection and screening.The miR-21 knockout efficiency was around 10/10 original cells.Sequencing the mutant clones indicated that miR-21 expression could be drastically reduced by simply altering sequences immediately adjacent to the microRNA duplex.Conclusion:This strategy may be applied to knockout any microRNA of interest even in hemapoietic cell lines with very low transfection efficiency.