MYCN转基因斑马鱼对造血调控因子的影响

The influence of MYCN gene on hematopoietic regulatory factors in MYCN transgenic zebrafish

目的:通过热激活启动子条件性过表达鼠源性MYCN基因,建立生殖系稳定转染的斑马鱼系,以研究MYCN基因对造血调控因子转录的影响。方法:构建携绿色荧光蛋白"报告基因"的MYCN质粒,显微注射到斑马鱼胚胎单细胞期,通过荧光筛选出F0代,继而建立生殖系稳定转染的转基因鱼系。通过反转录聚合酶链反应(RT-PCR)、实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)及血涂片论证MYCN基因的过表达及其对造血调控因子的影响。结果:在256条嵌合表达的性成熟F0代斑马鱼中,鉴定发现有18条(7.0%)显示转基因阳性,其中雄鱼8条,雌鱼10条。同时,RFQ-PCR及外周血涂片均显示,转基因斑马鱼F1和F2代均发生明显的造血分化障碍,造血关键调控因子scl、mpo、gata1基因表达量明显下降。结论:斑马鱼在基因研究中具有直观及规模化优势,特别适用于单一基因的研究,MYCN基因产物可显著抑制红系生成和造血分化。

Objective To establish stable germline MYCN transgenic zebrafish by conditionally overexpressing murine MYCN gene through heat-shock promoter for studying the influence of MYCN gene on transcriptional regulation of hematopoiesis.Methods MYCN plasmid carrying green fluorescent protein ＂reporter gene＂ was constructed and then microinjected into zebrafish single-cell embryos,and then identified the F0（founder） generation by fluorescence screening for establishing the zebrafish stable transgenic germline.Over-expression of MYCN gene and its influence on hematopoietic regulatory factors were assayed through RT-PCR,RFQ-PCR and blood smear.Results Eighteen of the 256（7.0%） mosaic F0 zebrafish embryos injected with the constructed vector were identified to be the germline transgenic zebrafish,including 8 males and 10 females.RFQ-PCR and blood smear showed obvious hemopoietic differentiation obstacle in transgenic zebrafish.Expressions of scl,mpo,gata1 mRNA,which were the key hematopoietic regulatory factors,were apparently decreased.Conclusions Zebrafish offers the advantages of direct visual and high-throughput scale for studying gene function,especially for a single gene.MYCN gene could inhibit significantly erythropoiesis and hematopoietic differentiation.