Establishmentandcharacterizationofa?broblast-likecelllinefromAnabariliusgrahami(Cypriniformes:Cyprinidae)

ThoughYunnanprovincecontainssome562knownspeciesoffish,nocelllinesfromanyofthesehavebeenmadeavailabletodate.ToprotectgermplasmresourcesandprovideaneffectivetoolinsolvingproblemsatcellularlevelofAnabariliusgrahami,afishendemictoFuxianLake,Yunnan,China,weestablishedandcharacterizedthemajorfeaturesofacontinuouscellline(AGFII)fromthecaudalfintissueofA.grahami.ThisAGFIIcelllineconsistsoffibroblast-likecellsandhasbeensubculturedmorethan60timesoverthecourseofayear.ThecelllinewasmaintainedinDMEM/F12supplementedwith10%FBS,withacellulardoublingtimeof51.1h.Wecontinuedwithmoreexperimentstooptimizethecultureandstorageconditions,andfoundavarietyofinterestingresults:cellscouldgrowattemperaturebetween24癈and28癈,withtheoptimaltemperatureof28癈.Likewise,thegrowthrateofA.grahamifincellsincreasedwhentheFBSproportionincreasedfrom5%to20%,withtheoptimalgrowthattheconcentrationsof20%FBS;cellswereabletogrowinL-15andDMEM/F12withoptimalgrowthatL-15;DMSOisabettercryoprotectantthanGlycerol,EGandMeOHforAGFIIcellswithoptimalconcentrationof5%DMSO.Chromosomeanalysisalsoshowedthatthedistributionofchromosomenumbervariesfrom38to52,withamodalpeakat48chromosomes,accountingfor39.8%ofallcells.UsingthesameprimerpairsspecifictomtDNA,theAGFIIcellsequencesobtainedbyPCRwereidenticaltothosefrommuscletissuesofA.grahami.BothchromosomeanalysisandPCRamplificationconfirmedtheAGFIIcellswerefromA.grahami,alsoindicatingthatthatcurrentlong-termartificialpropagationofA.grahamihasbeensuccessful.Finally,wenotedthatwhencellsweretransfectedwithpEYFP-N1andpECFP-N1plasmid,brightfluorescentsignalswereobserved,suggestingthatthiscelllinemaybesuitableforuseintransfectionandfuturegeneexpressionstudies.