SB转座子介导的斑马鱼增强子捕获注解分析

发布时间：2018-05-08 07:29

本文选题：增强子捕获 + 注解　；参考：《生物技术通报》2017年05期

【摘要】：为了建立斑马鱼增强子捕获突变体库及研究功能基因的表达调控模式,制备了SB转座子介导的增强子捕获转基因斑马鱼(F0),通过繁育建立了组织或器官特异表达报告基因绿色荧光蛋白(Green fluorescent protein,GFP)的品系(F1)。选择F1代的SK-3系(脑部特异表达GFP)与野生型TU系斑马鱼交配,收集受精卵(F2),于24 hpf(Hour post fertilization)、2 dpf(Day post fertilization)、3 dpf、4 dpf、5 dpf、7 dpf 6个发育阶段检测报告基因绿色荧光蛋白的表达模式;然后通过Splinkerette PCR(SP-PCR)方法检测SB转座子在斑马鱼基因组中的插入位置,从而确定捕获的增强子和功能基因;最后通过胚胎原位杂交验证内源基因表达模式。结果表明,在F2代胚胎不同发育阶段,GFP表达具有明显的时空特性,前期在前脑,中脑,后脑三个部位均高水平表达,后期表达部位呈后移趋势,各个发育期表达强度基本无变化,结果提示该捕获增强子具有脑部表达特性。sp-PCR结果分析表明增强子位于基因组1号染色体35、914、498-35、914、621位置,在内源性基因ednraa位点附近。原位杂交结果表明该基因在胚胎24 hpf阶段具有转录活性。本研究结果提示SB转座子介导的增强子捕获技术可高效获得插入突变斑马鱼,对研究基因功能和获得具有自主知识产权的新基因具有重要作用。
[Abstract]:In order to establish the catch mutants library of zebrafish enhancer and to study the expression and regulation of functional genes, The SB transposon mediated enhancer was prepared to capture transgenic zebrafish F0, and the strain F1 was established by breeding the tissue or organ specific expression reporter gene Green fluorescent protein (GFP). F1 SK-3 lines (brain specific expression GFPs) were selected to mate with wild type TU zebrafish. The F _ 2s of fertilized eggs were collected, and the expression patterns of green fluorescent protein (GFP) in 6 developmental stages of the report gene were detected at 24 hpf(Hour post fertilization-2 dpf(Day post development stage. Then the insertion position of SB transposon in zebrafish genome was detected by Splinkerette PCR- SP-PCR method to determine the captured enhancer and functional gene, and the expression pattern of endogenous gene was verified by in situ hybridization. The results showed that the expression of GFP in F _ 2 embryos at different stages of development had obvious spatio-temporal characteristics. The expression of GFP was highly expressed in the forebrain, midbrain and hindbrain in the Prophase, and the tendency of the expression was backward in the later stage. There was no change in the expression intensity in all developmental stages. The results showed that the enhancer had the characteristics of brain expression. SP-PCR analysis showed that the enhancer was located in the position of chromosome 1, 35914498-35914621, and located near the ednraa site of endogenous gene. The results of in situ hybridization showed that the gene had transcriptional activity at 24 hpf stage of embryo. These results suggest that SB transposon mediated enhancer capture technique can efficiently obtain inserted mutant zebrafish and play an important role in studying gene function and obtaining new genes with independent intellectual property rights.
【作者单位】：扬州大学动物科学与技术学院扬州大学农业与农产品安全国际合作联合实验室;
【基金】：国家自然科学基金项目(31671313,31572364) 扬州现代农业项目(YZ2016040)
【分类号】：Q78

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