Määritelmä
In1987,VleinfirstreportedtheapplicationofthistechnologytoadsorbTMV(tobaccomosaicvirus)RNAtothesurfaceoftungstenparticlesandbombardtheonionepidermis.Aftertesting,itwasfoundthattheviralRNAcouldreplicate,andtheCAT(Chloraphericolacetyltransferasegene)genewasintroducedintoonionepidermalcellsusingthesametechnique.Thetechnologyhasbeensuccessfullyappliedtotobacco,rice,wheat,ryegrass,sugarcane,cotton,soybeans,kidneybeans,onions,papaya,sweetorange,grapesandothercrops.
Geeniaseiden luokitus
Thismethodreliesonakindofgeneguntohelpintroduceforeigngenes.Accordingtothepowersystem,thegeneguncanbedividedintothreecategories:gunpowderdetonation,highvoltagedischargeandcompressedgasdrive.Thebasicprincipleisthatthemetalparticles(goldortungstenparticles)withgenesareadsorbedonthesurfacebythepowersystem,andtheDNAisinjectedintotheplantcellsatacertainspeed.Becausethesmallparticleshavestrongpenetratingpower,thereisnoneedtoremovethecellwall.Andcellmembranetoenterthegenome,soastoachievethepurposeofstabletransformation.Ithastheadvantagesofwideapplication,simplemethod,shortconversiontime,highconversionfrequency,andlowexperimentcost.ForplantsthatcannotbeinfectedbyAgrobacterium,thismethodcanbreakthelimitationsofthevectormethod.Thefrequencyofgeneguntransformationisdirectlyrelatedtothetypeofreceptor,thesizeofthemicroprojectile,thebombardmentpressure,thedistancebetweenthestopdiscandthegoldparticles,thepretreatmentofthereceptor,andthecultureafterthebombardmentofthereceptor.
Asemenetelmä
Genegunmethod:Soak4umdiametertungstenpowderorgoldpowderinthedonorDNA,andthenusethegeneguntoinjecttheseparticlesintocells,tissuesororgans.Ithastheadvantageofprocessingmultiplecellsatonce,butthetransformationefficiencyislow.Inaddition,thismethodisalsousedingenetherapyandantibodypreparation,andhasachievedpreliminaryresults.
Turvallinen menetelmä ja vaikutus
Genegun,alsoknownasparticlebombardment,high-speedparticlejettechnologyorgenegunbombardmenttechnology,wasdevelopedbyJohn.C.,DepartmentofBiochemistry,ComelUniversity,USASantordisequaltothesuccessoftheresearchin1983.First,itwassuccessfullyappliedinplants.Throughthepowersystem,themetalparticles(goldortungstenparticles)withgenesareadsorbedonthesurface,andtheDNAisinjectedintothetargetcellatacertainspeedtoachievethepurposeofstabletransformation.Smallparticleshavestrongpenetratingpoweranddonotneedtomodifytargetcells.Genegunhastheadvantagesofwideapplication,simplemethod,non-strictrequirementonthesizeoftherapeuticgene,shortconversiontime,longtransientexpressionduration,processingmultiplecellsatatime,andhighsafety.UsingDNAthatis2to3ordersofmagnitudelowerthantheordinaryinjectionmethodcanproducehigherprotection,butthetransformationefficiencyisrelativelylow.Itisawidelyusedandveryefficientimmunemethod.
In2003,somescholarsproposedthatgenegunimmunizationissuperiortointramuscularinjectionregardlessoftheantibodytiteritmediatedandtherequiredDNAvaccinedose.Inordertoprovethisstatement,KimHJetal.clonedtheAMA-1geneofP.vivaxandexpresseditintheplasmidvectorUBpcAMA-1toobtainaproteinofapproximately56.8kDa.BALB/cmicewereimmunized4timesbyintramuscularimmunizationorgenegun,andtheproportionofsplenicTcellsubtypeswasmeasuredbyflowcytometry2weeksafterthelastinjection.TheexperimentalresultsshowedthattheintramuscularlyinjectedmousespleencellshadnosignificanceintheratioofCD8+TcellstoCD4+Tcells;however,asignificantincreasewasobservedinmiceimmunizedwithgenegun(P<0.05)CD8+cells.TheresultsshowedthatwhentheplasmidDNAvaccineencodingP.vivaxAMA-1wasinjectedintramuscularly,theresultingcellularimmunogenicitywasverylow;however,significanteffectscouldbeobservedbythegeneguninjectiontechnique.
TransferofDNAvaccinewrappedwithgoldparticlesviageneguncaninhibitthegrowthoftumortissues.AbeAetal.usedintradermalgeneguntotransferDNA(pNGVL-hFLex)wrappedgoldparticlestothemouseskinaroundthetargettumor,anddetectedaFlt3ligandFL(anewlydiscoveredcytokine,whichismediatedbygenegun).Promotethegrowthofdendriticcells)Theinhibitoryeffectofmetastasisontumorgrowthinamousemodel.Experimentalresults:Immunohistochemistryandfluorescenceactivatedcellclassification(FACS)showedthatgenegun-mediatedDNA(pNGVL-hFLex)transfersignificantlyincreasedthenumberofCD11c(+)DCsintumortissuesandinhibitedMCA205tumorsGrowth.ItcanbeconcludedthatthesuccessfulFLtreatmentthroughgenegunsignificantlyincreasedthenumberofDCsinthetumortissueandinhibitedthegrowthofthetumor.
Duetothehighcostofgoldpellets,ChenCadetal.comparedtheimmuneeffectsproducedinthebodywhennakedDNAvaccinesandDNAvaccineswrappedingoldpelletsweretransportedthroughlow-pressuregenegunsandhigh-pressuregeneguns,respectively.Theexperimenttestedwhetherthenon-carriernakedDNAvaccinedeliveredbylow-pressuregeneguncanproducesimilarantigen-specificimmuneresponseandanti-tumoreffectcomparedwiththeDNAvaccinewrappedwithgoldbeads.Theresultsshowedthat:inmice,nakedCRT/E7DNAvaccineresultedinastrongerimmuneresponse,withasubstantialincreaseinE7-specificCD8+TcellprecursorsandE7-specificantibodies;nakedCRT/E7DNAvaccineproducedastrongAnti-tumoreffectagainstsubcutaneousE7expressingtumorsandagainstmetastaticlungcancerbeforeE7expression;Inaddition,miceimmunizedwithnakedCRT/E7DNAvaccineareontheskinsurfacecomparedwithmiceimmunizedwithCRT/E7DNAvaccinewrappedwithgoldbeadsTheimpactofburnsissignificantlyreduced.Theexperimentfinallyconcluded:ThenakedCRT/E7DNAvaccineistransportedbylow-pressuregenegun.Comparedwiththetraditionalgoldbead-wrappedDNAvaccine,itcanproduceasimilarstrongimmuneresponseandeffectiveanti-tumoreffect,anditismoreconvenientandhasasmallersize.Sideeffects.