The Generation Of Transgenic Marine Medaka(Oryzias Melastigma)Varieties For Ornamental Applications By Tol2 Transposon System

The ornamental fish industry is quite promising due to its advantages of low water consumption,less land requirements,technology-intensive and high yield.According to the estimates of Food and Agriculture Organization(FAO),the annual sales of global ornamental fish market is expected to about six-billion-dollar,and the gross annual output of other related industry is expcected to about fifteen-billion-dollar.Recently,the ornamental fish industry has changed from traditional stock model to a more refinement,and artistic model.Meanwhile,aquarium fish genetically modified to fluoresce in different bright colours has opened a new market and brought ample opportunities.Now,three companies,Yorktown Technologies,Taikong Corp and Jy Lin Trading Company,focus on transgenic fish,but only on freshwater fish.Marine medaka(Oryzias melastigma)is also known as Indian medaka,belongings to beloniformes,adrianichthyidae,oryzias.The fish body is nealry transparent and the body length is about 3.5-4 cm.Due to the strong environment tolerance,it is easy to rear this species on a large scale culture under the laboratory environment.Therefore,the present study is to transgenic marine medaka varieties for ornamental applications by Toll transposon system.The results are shown as below:(1)One factor influencing the microinjection procedure is the thickness of O.melastigma egg chorion(outer envelope).Therefore,we first generated glass needles that were strong enough to penetrate extremely thick and robust chorion of O.melastigma eggs,and thin enough to allow injection of solutions into the cytoplasm of the egg at one-cell stage.By using Tol2 transposon system,the foreign DNA fragments:efla-eGFP(green fluorescent protein)or efla-mVenus(yellow fluorescent protein),were integrated into the genome of O.melastigma.We observed the mosaic eGFP pattern in founder fish.By crossing with wild type,we get the F1 fish which showed all cells expressing fluoresce proteins.EGFP expression in the F1 fish varied in intensity,and the green fluorescent colour of the strongest intensity individuals can be seen by the unaided eyes under the aquarium blue light conditions,which makes ornamental value.(2)We cloned 1084 base pair(bp)promoter of myosin light chain 2 gene(mlc2f)of zebrafish,then builded the pminiTol2-mlc2f-mCherry vector by ligatingthree DNA fragments:mlc2f promoter,mCherry(red fluorescent protein)gene,pminiTol2 vector backbone.Using Tol2 transposon system,we successfully integrated foreign DNA fragment:mlc2f-mCherry into the genome of O.melastigma.We observed the mosaic mCherry pattern in founder fish.By crossing with wild type,we get the F1 fish which showed muscle cells expressing mCherry.This transgenic lines produced a strong and vivid red-purple fluorescent color that was easily seen with the unaided eye udner normal daylight condition.(3)The 2011 bp promoter sequnce of actinodinl(andl)gene from O.melastigma was cloned.The pminiTol2-andl-eGFP vector was builted by ligating DNA fragments of andl promoter,eGFP gene and pminiTol2 vector.By using Tol2 transposon system,the foreign DNA fragment:andl-eGFP was integrated into the genome of O.melastigma.In F1 transgentic fish,the eGFP were expressed in both actinotrichia and lepidotrichia.More interestingly,muscles closed to basement of fins expressed eGFP as well.Unfortunately,this transgenic lines exhibited a weak fluorescent color that impossiblly be seen with the unaided eye udner normal daylight condition.In summary,we have successfully built four different transgenic marine medaka varieties,and the mlc2f-mCherry line has a highly brilliant red-purple fluorescent color over whole skeletal muscles,which might be sufficient for ornamental application.The results from the present study indicated that other varieties of transgenic marine medaka lines can be built under the promoter of mlc2f together with other fluorescent protein gene.