斑马鱼心肌特异性启动子的克隆及其功能鉴定*

目的:探讨心室肌球蛋白重链(vmhc)基因启动子的心肌组织特异性.方法:利用PCR技术从斑马鱼基因组中克隆了vmhc编码区5’上游大小为1952bp的调控区域,应用酶切连接方法将vmhc启动子插入pGEFP-N1质粒,成功构建pEGFP-vmhc重组载体.再应用高保真DNA聚合酶PCR扩增包含vmhc启动子序列,增强型绿色荧光蛋白(EGFP)基因序列及3'UTR序列的基因片段,经过纯化后通过显微注射将vmhc-EGFP基因片段导入斑马鱼受精卵中.结果:注射后的斑马鱼心脏中出现绿色荧光,而其他部位无荧光出现.结论:vmhc启动子能够正确有效地驱动外源基因在斑马鱼心脏中特异表达,适合应用于心血管疾病的基因功能研究,基因靶向治疗等.

Cloning and Functional Identification of the Zebrafish Cardiac-specific Promoter*

Objective: To study the cardiac-specific expression of ventricular myosin heavy chain(vmhc) gene promoter.promoter.Methods: A 1952 bp promoter fragment of zebrafish vmhc gene 5’end deletion upstream region was amplified by PCR from zebrafish.Vmhc promoter was inserted into pGEFP-N1plasmid by enzyme digestion to construct the recombinant vector of pEGFP-vmhc.The gene fragment contain vmhc promoter sequences,enhanced green fluorescent protein gene(EGFP) sequences and 3’UTR sequences was amplified by PCR with high-fidelity DNA polymerase.The purified vmhc-EGFP gene fragment was injected into fertilized eggs of zebrafish by Microinjection.Results: Green fluorescence could be observed in the zebrafish heart injected the vmhc-EGFP gene fragment,but not in Non-myocardial tissue.Conclusion: Vmhc promoter is able to correctly and effectively drive the specific expression of exogenous genes in the zebrafish heart,and it is suitable for the study of gene function and gene therapy in cardiovascular disease.