A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse 英文参考文献.docVIP

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A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse 英文参考文献.doc

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A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse 英文参考文献

ASimplePCRMethodforRapidGenotypeAnalysisofthe TH-MYCNTransgenicMouse SeikiHaraguchi1,AkiraNakagawara2* 1LaboratoryofEmbryonicandDevelopmentalGenetics,ChibaCancerCenterResearchInstitute,Chuo-ku,Chiba,Japan,2DivisionofBiochemistryandInnovativeCancer Therapeutics,ChibaCancerCenterResearchInstitute,Chuo-ku,Chiba,Japan Abstract Background:TheTH-MYCNtransgenicmouseisthemostwidelyusedmurinemodelofhumanneuroblastoma,inwhicha human MYCNoncogene is targetedtoneuroectodermal cells of developingmice under the influenceof therat tyrosine hydroxylasepromoter.Sofar,homozygoustransgenicmicehavebeenidentifiedbyeitherSouthernblotorquantitativereal- timePCR. Principal Findings: To establish a simple and reliable genotyping method by conventional PCR, we confirmed the integration of the transgene in the TH-MYCN transgenic mouse by Southern blot and inverse PCR analyses. Our results showedthateitherfiveorsixcopieswerefoundtobeinsertedinahead-to-tailtandemconfigurationatasinglelocus.The MYCNtransgene/hostDNAjunctionwassequencedandtheintegrationsitewasidentifiedatchromosome18qE4.Finally, we succeeded in designing rapid, simple and reliable genotyping method by common PCR using primers flanking the integratedTH-MYCNtransgene. Conclusion:WeestablishedasimpleandreliablegenotypingPCRmethodfordeterminingtheintegrationsiteoftheTH- MYCNtransgenethatenablesallpossiblegenotypestobedistinguishedwithinseveralhours.TH-MYCNmiceareexcellent model for human neuroblastoma study, thus our results will largely be useful for facilitating thepace of neuroblastoma study,includinginthestudyofthetumourigenicprocess,andinthedevelopmentoftherapiestotreatpatientssuffering fromneuroblastoma. Citation: Haraguchi S, Nakagawara A (2009) A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse. PLoS ONE 4(9): e6902. doi:10.1371/journal.pone.0006902 Editor:EdatharaAbraham,UniversityofArkansasforMedicalSciences,UnitedStatesofAmerica ReceivedMay21,2009;AcceptedJuly7,2009;PublishedSeptember4,2009