荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

Construction of fluorescent transgenic zebrafish Tg (ttn.2: EGFP)

摘要:

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein, EGFP)转基因斑马鱼品系，本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体，并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎，通过荧光检测、遗传杂交筛选和分子鉴定等方法，成功建立了能稳定遗传的Tg (ttn.2: EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明，绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点，结果表明：No. 33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上，No. 34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg (ttn.2: EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外，绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。

Abstract:

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2:EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2:EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.