幼年新西兰白兔损伤关节软骨再生相关基因cdna差示文库构建及部分相关基因分析

《幼年新西兰白兔损伤关节软骨再生相关基因cdna差示文库构建及部分相关基因分析》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、第三军医人学硕+学位论文diameterand3mmindepth，penetratingthesubchondralboneplate，wascreatedonthemedialfemoralcondyleasfartotherearaspossible，usingabit．A1ltherabbitswerekilledfourweeksafteroperationwiththemethodofaeroembolism．Immediatelyafterdeath，bothhindlimbsweredisarticulatedatthehipjoin

2、tandtherepairedtissuewereseparatedandstoredattheliquidnitrogen．ThetotalRNAoftheyongandtheadultrabbit’srepairedtissuewasextractedandpreparedasthetemplateofRT—PCR．ThecDNApreparedfromtheyongrabbitwasusedasthetesterandthecDNAfromadultrabbitasthed“ver．ThedstesterandthedrivercDNAwasd

3、igestedintoshorter，blunt—endeddscDNAfragmentswithRsaI．ThetestercDNAwasaliquottedintotwoparts．OnealiquotwasligatedwithAdaptor1andthesecondwasligatedwithAdaptor2R．ThentwohybridizationsandtwoPCRamplificationswereperformed．ThePCRproductioncontainingdifferentiaUyexpressedcDNAswaslig

4、atedwithTVectorandwastranslatedintocompetentEc01i．ThepositiVecolonieswereselectedbyblue—whiteexperimentandthedifferentiallibrarywasconstructed．ThepositiVecoloniesweresequencedafteridentificationbyPCR．TheESTsofthedifkrentiallyexpressedgeneswereobtainedwiththesoftwareDNAStar5．01a

5、ndthesoftwareChromasandanalysisedbytheReVerseNorthernblot．BLASTNtheESTsequencesinGENEBANKandselectedtheaimedgenef}agmentsasthefbrtherresearchingtarget．Results：ThetotleRNAextractedthroughone—stepmethodcombiningplantRNAoutkithadahighqualityandwasreVerselytranscriptedintodscDNAbyL

6、D—PCR．Thedif色rentiallibraryofyongrabbit’repairedtissuewassuccessfUllyconstructedbysuppressionsubtractedhyb“dization(SSH)techn0109yandSwitchingMechanismAt5’endoftheRNATranscript(SMART)techn0109y．Weobtained223indiVidualcDNAclones，and64ofthemwereexaminedbyPCR．Twentydiffrentialexpr

7、essedgenefragmentswereobtainedaftersequencingandanalysisingwithsoftware，13ofthemwereidentifiedbyhomologysearch(including1Vectorsequenceand6nonsensesuquencessuchasmitochondrialgene，ribosomalgeneetc．)and6ofthemwereunreported．Dotblottestifiedthat18of20werethedifferentialexpressedg

8、enesand2of20wasexcluded．Inthetestified18geneftagments，