Regarding the uncertainty of the exact cause of the acute lymphocytic leukemia (ALL) caused by ETV6-RUNX1t(12;21) translocation, correcting genes of the ETV6 and RUNX1 in ETV6/RUNX1 fusion gene simultaneously on chromosome 12 may be effective in reducing leukemia malignancy. Thus, we designed an homologous recombination (HR) plasmid to target of the ETV6/RUNX1 fusion gene in the REH cell line containing the ETV6-RUNX1t(12;21) translocation. Cells were cultured and transfected by HR plasmid. The presence of the replacement cassette at specific location in the ETV6/RUNX1 fusion gene was verified by PCR and sequencing method. A quantitative gene expression assay was performed to evaluate changes in expression of ETV6, RUNX1, and ETV6/RUNX1 genes following editing. The cell viability was measured by trypan blue staining. The expression of the ETV6 gene was significantly increased in modified cells than unmodified cells by 10.9-fold. In contrast, the expression of the ETV6-RUNX1 fusion gene was significantly decreased in the modified cells compared with unmodified cells by 0.26-fold. The expression of the RUNX1 gene had no significant difference between modified and unmodified cells. The survival rate of edited cells was significantly decreased than unedited cells (p = 013). We designed a gene targeting system based on HR method to correct genes of ETV6 and RUNX1 simultaneously in ETV6/RUNX1 fusion gene on chromosome 12 containing ETV6-RUNX1t(12;21) translocation. The modification of this translocation may lead to reducing effects of the fusion gene’s damaging and the dosage compensation related to ETV6 and RUNX1 genes and subsequently reduce the effects of leukemia. This targeting system may open a window for treating leukemia as ex vivo.

中文翻译：

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急性淋巴细胞白血病细胞中ETV6/RUNX1易位的校正：通过同源重组机制的新基因靶向系统。


鉴于ETV6-RUNX1t(12;21)易位引起的急性淋巴细胞白血病(ALL)确切病因尚不确定，同时纠正12号染色体上ETV6/ RUNX1融合基因中的ETV6和RUNX1基因可能有效减少白血病的发生恶性肿瘤。因此，我们设计了一个同源重组 (HR) 质粒，以包含 ETV6-RUNX1t(12;21) 易位的 REH 细胞系中的ETV6/RUNX1融合基因为目标。培养细胞并用HR质粒转染。通过PCR和测序方法验证了ETV6 / RUNX1融合基因中特定位置处替换盒的存在。进行定量基因表达测定以评估编辑后ETV6 、 RUNX1和ETV6/RUNX1基因的表达变化。通过台盼蓝染色测量细胞活力。修饰细胞中ETV6基因的表达比未修饰细胞显着增加10.9倍。相反，与未修饰的细胞相比，修饰的细胞中ETV6-RUNX1融合基因的表达显着降低0.26倍。 RUNX1基因的表达在修饰和未修饰的细胞之间没有显着差异。编辑细胞的存活率显着低于未编辑细胞（ p = 013）。我们设计了基于HR方法的基因打靶系统，对含有ETV6-RUNX1t(12;21)易位的12号染色体上的ETV6/RUNX1融合基因中的ETV6和RUNX1基因同时进行校正。 这种易位的修饰可能会导致融合基因损伤和与ETV6和RUNX1基因相关的剂量补偿的影响减少，从而减少白血病的影响。这种靶向系统可能为离体治疗白血病打开一扇窗。