绿色荧光蛋白基因向花鲈胚胎的转移及其表达

Expression of green fluorescent protein gene transferred into newly fertilized sea perch (Lateolabrax japonicus) embryos by microinjection

通过显微注射的方法,将绿色荧光蛋白(Greenfluorescentprotein,GFP)基因注射入花鲈(Lateolabraxjaponicus)单细胞期受精卵动物极细胞质内。初步研究了显微注射后的花鲈胚胎的存活状况。在荧光显微镜下观察到了GFP的表达,多数胚胎表现为嵌合性表达。利用PCR技术,从花鲈尾芽期胚胎DNA中扩增出了特异片段,大小为750bp,表明显微注射胚胎中整合了外源GFP基因。

Fish are suitable types of animals to use in terms of introduction of novel genes into their genomes. Transgenic fish have many potential applications in aquaculture. Many researchers have done a lot about transgenic fish in order to improve their importantly commercial phenotypes, such as growth, disease resistance, freeze resistance. A lot of methods are used to introduce novel genes to fish genomes and microinjection is a leading methods. Sea perch Lateolabrax japonicus is one of the most important species in the aquaculture of China. In this article, plasmid pCMVGFP containing green fluorescent protein (GFP) genes was injected into the cytoplasm of newly fertilized sea perch embryos at one cell stage. An appropriate volume of fifty picoliters of DNA solution containing 10~5～10~6 moleculars was introduced into every egg. The mortality of both injected and uninjected embryos increased markedly in the first two days after impregnation and then increased unmarkedly. The mortality of injected embryos was higher than that of uninjected embryos at every period. The survival rate of injected embryos was only 3% after 5 days while that of uninjected embryos was 39%. A pair of primers was synthesized to detect whether novel GFPgenes were integrated into sea perch genome by polymerase chain reaction(PCR) technology. A specific GFP segment of 750 bp was amplified from the DNA of sea perch embryos at tail-bud stage, and three groups showed positive. This specific segment was also amplified from the genome DNA of sea perch five-day-old larvae, and showing that exogenous GFP genes were integrated into three larvae genomes. We observed the expression of GFP in the sea perch embryos through a fluorescence microscope. GFP was expressed largely after 48 hours. Embryos that expressed GFP decreased with the development. The expression was mostly mosaic. The embryos that expressed GFP althrough the bodies were observed before hatch, but the larvae that expressed GFP partly in the bodies were observed after hatch.