Exogenous Genes Construction, Transfer In To Embryos Of Zebrafish (Dania Rerio) And Sea Perch (Lateolabrax Japonicus)

Fish are very suitable types of animals to use in terms of the 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 were used to introduce novel genes to fish genomes and microinjection is a leading methods.In this article, plasmid pCMVnramp was constructed using plasmid pCMVGFP and Nramp cDNA. Nramp gene is driven by CMV promoter. And different Exogenous genes including GFP gene, Nramp gene and Magainin gene were injected into the cytoplasm of newly fertilized sea perch and zebrafish embryos at one cell stage. An appropriate volume of 50 picoliters of DNA solution containing 105~106 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. Expression of GFP was observed in the sea perch and zebrafish embryos through a fluorescence microscope. GFP was expressed largely after 48 hours. Then embryos that expressed GFP decreased with the development. The expression was mostly mosaic. Embryos expressing GFP throughout the bodies were observed before hatch, but the larvae that expressed GFP partly in the bodies were observed after hatch. Three pairs of primers were synthesized to detect whether exogenous genes were integrated into sea perch genome by polymerase chain reaction(PCR) technology. A specific GFP segment of 750bp was amplified from the DNA of sea perch embryos at tail-bud stage. This specific segment was also amplified from the genome DNA of sea perch five-day-old larvae, but the segment from zebrafish 3-day-old larvae was feeble. The results showed exogenous GFP genes were integrated into larvae genomes. No specific segment was amplified from genome of sea perch embryos injected with Magainin gene. A specific 1700bp segment of Nramp was amplified from genome DNA of zebrafish 3-day-old larvae.