1、Y773620分類扎密級：絮目莠≮夭等博士學位論文單位代碼：10019學號；B02311小尾寒羊骨骼I]fLgN]包Myostatin基因打靶的研究MyostatingenetargetinginculturedHanyangovineskeletalmuclecells研究生：指導教師：申請學位門類級別：專業(yè)名稱：研究方向：所在學院：韭萱醫(yī)丞揖麴援理堂擅I：生理堂動物基國蘭猩生物堂瞳二零零五年六月AbstractGenetargeti

2、ngisatechniquethatintegratesexogenousgoneintotheexpectedsiteofcellularchromosomebyhomologousrecombinationtochangecelloranimal’sheritagecharactersThispaperreportallinvestigationdesignedtoknockoutmyostatingenebygonetargeti

3、nginHanyangovineskeletalmusclecellsTwopromotertraptargetingvectorsMSTNGFPandMSTNNeowereconstructedandwereusedtotransfectfetalandnewbornovineprimaryskeletalmusclecellsBothGFPexpressingcellsanddrugresistantcellswereobtaine

4、datalloveralltargetingefficiencyof6x10～G418resistantcellswerecharacterizedbyPCRandSouthernbloUingaftergrowingintocellclones，Myostatin(MSTN)amemberofthetransforminggrowthfactorB(TGF—B)superfamilyhasbeenshowntobeanegativer

5、egulatorofmyogenesisTheexistingofararemultiplebirthHanyangsheepinChinaencourageustoexplorethepossibilityofdownregulationoftheactivityofmyostatingenebygonetargetingwherebyasheepwithmeritsofbothmultiplebirthandwell—develop

6、edmusclemightbecreatedA50kband10kbMSTNgonefragmentwasobtainedbylongPCRfromgenomicDNApreparationsandwasclonedintopMD18TvectorrespectivelyThepromotertrapvectorMSTNNeocompriseda50kbfragmentoftheMSTNgonecontainingexonl，intro

7、nl，exon2，inon2andaportionofexon3Thelonghomologousam]was46kbfromthe50kbfragment；an10kbfragmentcontainingaportionofexon3andthe3’endregionoftheMSTNgoneTheshorthomologousarnlwas08kbofthe10kbfragmentTheskeletalbacterialclonin

8、gvectorwaspGEM3zf()TheselectionmarkergenewereNeoandGFPrespectivelyThesetwopromterlesstargetingvectorwereconstructedbyinsertingsuccessivelythethreeDNAfragmentsintothecloningvectofLinearvectorDNAmoleculeswereproducedbycutt

9、ingthevectorattheuniqueAtllsiteandwereusedfortransfectionofovineskeletalmusclecellsincultureThevectorMSTNGFPwasconstructedinasimilarwayexce：ptthattheselectionmarkerNeowasreplacedbyGFEPrimaryHanyangovineskeletalmusclecell

10、swereisolatedfromthehindlimbofafiftydaysoldmalefetusandofaoneweekoldlambrespectivelyThemuscletissuewasdisaggregatedbytrypsinsolutionandthecellswereculturedandpassagedinvitroOvineskeletalmusclecelllinewasconstructedTransf

11、ectingprimarynewbornovineskeletalmusclecellswithMSTNGFPusinglipofectAMINEaccordingtothemaufacturer’sprotoc01TheexpressionofGFPincellswasconfirmedbyobservingthegreenfluorescence24hsaftertransfectionWecoulddetected，onavera

12、ge，23fluorescentcellsper103recipientcellsaftertransfectionandgrowthfor24hours48haftertransfectionovineskeletalmusclecellsandfetalskeletalmusclecellswithMSTNNeo，G418selectionwasappliedfor610daysForthecomparisonoftheeffect

13、ofserumondrugselection15％FCSand10％FCSwereusedintheculturemediumAtotalof88NeoresistantcoloniesweregeneratedHomologousrecombinationeventsweredetectedbyPCRamplificationandSouthernblotting，onlyoneclonewasconfirmedthatonealle