AML1-EVI1转基因斑马鱼模型的建立及对造血转录因子的调控

The establishment of AML1-EVI1 transgenic zebrafish model and regulation of hematopoietic transcription factors

目的:通过建立表达急性髓系白血病(AML)1-同向性病毒整合位点(EVI)1融合基因斑马鱼模型,研究AML1-EVI1融合基因对造血转录因子的影响。方法:构建携带绿色荧光蛋白的AML1-EVI1质粒,显微注射到斑马鱼胚胎单细胞期,建立生殖系稳定转染的转基因模型,常规养殖到F2代。以F2代转基因斑马鱼作为实验对象,运用反转录(RT)-PCR验证AML1-EVI1融合基因的表达,外周血涂片、肾细胞涂片检测血细胞形态学改变,全胚胎原位杂交及荧光实时定量PCR(RFQ-PCR)检测融合基因对造血转录因子[GATA结合蛋白1(GATA1)、髓过氧化物酶(MPO)]的影响。结果:在性成熟146条嵌合表达的F0代斑马鱼中鉴定得到3条(2.1%)转基因阳性斑马鱼,其中雄鱼2条,雌鱼1条。外周血涂片及肾细胞涂片显示转基因斑马鱼出现造血细胞分化障碍,幼稚细胞增多,全胚胎原位杂交及RFQ-PCR均显示,与野生型相比,转基因型斑马鱼造血转录因子MPO基因表达量无论造血细胞发育早期还是晚期都明显下降,转录因子GATA1则在发育早期上调,晚期呈现明显下调。结论:AML1-EVI1融合基因通过调节转录因子抑制髓系分化,红系发育,促进原始细胞增殖。

Objective To study the influence of acute myeloid leukemia （AML）l-ecotropic virus integration site （EVI） 1 fusion gene on the transcriptional regulation of hematopoiesis by establishment of AML1-EVI1 transgenic zebrafish model. Methods AML1-EVI1 plasmid with reporter gene carrying enhanced green fluorescent protein （EGFP） was constructed. Then the vector was microinjected into single-cell zebrafish embryos to establish the model of stable germline transgenic zebrafish line,which was bred to F2 generation. AML1-EVI1 fusion gene in transgenic zebrafish was detected by RT-PCR. Blood cell morphology was detected by peripheral blood smear and kidney cell smear, and the influence on hematopoietie regulatory factors [globin transcription factor 1 （GATA1）, myeloperoxidase （MPO）] was assessed by whole- mount in situ hybridization and real time quantitative PCR （RFQ-PCR）. Results Three of 146（2.1%） mosaic F0 zebrafish embryos were identified as the positive germline transgenic zebrafish, including 2 males and 1 female. With F2 generation of transgenic zebrafish as experimental object, the morphology of peripheral blood smear and kidney cell smear showed the presence of blast cells and disturbance in differentiation. Whole-mount in situ hybridization and RFQ-PCR displayed that the expression level of MPO gene in the transgenic group was markedly decreased, and the expression level of GATA1 in the transgenic group was increased in the early stage then decreased in the late stage of hematopoiesis in transgenic zebrafish. Conclusions AML1-EVI1 fusion gene could significantly inhibit the erythropoiesis, suppress the myelocyte differentiation and promote primitive cell proliferation by regulation of transcription factors.