30393.重組酶刪除載體的構建及其在動物細胞中的應用

1、東北農(nóng)業(yè)大學碩士學位論文重組酶刪除載體的構建及其在動物細胞中的應用姓名：汪亮申請學位級別：碩士專業(yè)：動物遺傳育種與繁殖指導教師：劉娣20090620東北農(nóng)業(yè)大學農(nóng)學碩士學位論文IIConstructrecombinasevectapplicationintheanimalcellsAbstractWiththedevelopmentapplicationoftransgenictechnologydespiteitsgreatpoten

2、tialbenefititsnegativeeffectsgraduallyattractmemeattention..Asaimptanttechnologyfprolonginghumanlifespantransgenicanimalsfgantransplantationattractedawidespreadattentionhoweveritssafetyshouldneverbeneglected.Pigisanimpta

3、ntanimalmodelfgantransplantationresearchfthegreatsimilaritybetweenhumanpiginphysiology.ThepresentresearchesfocusonthedongandonanimalsofSwinepcinekidneybyitsparticularity.Afterobtainthecloningproducts，theproblemthattheexo

4、genousgeneespeciallymarkerreptergeneshouldbesaveddismissedattractsmanyresearcher’sresearchinterest.Itsnecessarytotheexogenousgeneindertominimizetheriskofpotentialfromthemarkerreptergenes.Thisexperimenttrytoconstructaplas

5、whichcontainstherecombinaserecognitionsitesbyusingthesitespecificrecombinationsystemtoverifythedeletioneffectsinanimalcells.Themainresultsincludes:（1）Basedonthetestrequiredwedesignedasequencethatcontainsthemultipleclonin

6、gsitesinthedlethedoublerecombinase(CreFLP)recognitionsiteonbothendobtainedthetargetfragmentwith211bpfulllengthbyoverlapextensionPCR(SOEPCR)thenthetargetfragmentwasclonedintopMD18TSimplebackbonevect（2）TakingpCDNA3.1pEGFPN

7、3pIRESplasasatemplatemarkerreptergeneswereamplified:includingneomycinresistance（neomycin）geneEnhancedgreenfluescentprotein（EGFP）genestrongpromoter(CMV)genethesequenceofinternalribosomeentrysite(IRES)（3）thetwomarkerrepter

8、genes（NeomycinEGFP）wereconnectedtothebackbonevectfexpressiondifferentproteinrespectivelycoexpressionofthetwoproteinbyIRESsystem.（4）Asthetwovectbackbonesfromthestep(3)wetheCrerecombinaserecognitionsiteobtainanothertwodiff