利用Tol2转座子构建斑马鱼心脏组织特异表达转基因载体及其表达分析

Construction and expression analysis of the zebrafish heart-specific transgenetic vector based on Tol2 transposable element

摘要:

为了制备用于在斑马鱼心脏中特异表达目的基因的转基因载体，通过分子克隆的方法对能够在斑马鱼心脏中特异表达EGFP报告基因的Tol2载体进行了改造，在原有的CMLC2启动子与EGFP编码区之间插入带有多克隆位点的IRES序列，获得pTol2-CMLC2-IRES-EGFP转基因表达载体，该载体可以实现在同一个启动子CMLC2的驱动下分别同时表达目的基因和EGFP；为了验证该表达载体的有效性，进一步在CMLC2启动子与IRES序列之间插入DsRed-Monome编码区，利用得到的pTol2-CMLC2-RED-IRES-EGFP转基因载体显微注射到斑马鱼单细胞期胚胎中进行表达分析，结果表明外源目的基因DsRed-Monome和报告基因EGFP均能以相同的表达模式在斑马鱼心脏组织中特异表达。pTol2-CMLC2-IRES-EGFP转基因表达载体的成功构建对于建立心脏发育候选基因的斑马鱼转基因实验模型具有重要意义。

Abstract:

In an effort to generate a desired expression construct for making heart-specific expression transgenic zebrafish, a Tol2 plasmid, which can drive EGFP reporter gene specifically expressed in the heart, was modified using subcloning technology. An IRES fragment bearing multiple cloning site (MCS) was amplified directly from pIRES2-EGFP plasmid and was inserted between the CMLC2 promoter and EGFP fragment of the pDestTol2CG vector. This recombinant expression plasmid pTol2-CMLC2-IRES-EGFP can drive any interested gene specifically expressed in the zebrafish heart along with EGFP reporter gene. To test the effectiveness of this new expression plasmid, we constructed pTol2-CMLC2-RED-IRES-EGFP plasmid by inserting another reporter gene DsRed-Monome into MCS downstream of the CMLC2 promoter and injected this transgenic recombinant plasmid into one-cell stage embryos of zebrafish. Under fluorescence microscope, both the red fluorescence and the green fluorescence produced by pTol2-CMLC2-RED-IRES-EGFP were detected specifically in the heart tissue in the same expression pattern. This novel expression construct pTol2-CMLC2-IRES-EGFP will become an important tool for our research on identifying heart development candidate genes’ function using zebrafish as a model.