科研设计-细胞生物学研究思路

细胞生物学研究思路,李家大医学遗传学国家重点实验室,研究背景,Waardenburg综合征（Waardenburgsyndrome，WS）是最常见的综合征型遗传性耳聋，主要遗传方式是单基因致病的常染色体显性遗传伴不全外显。主要临床特征：耳聋与着色异常（虹膜、皮肤、毛发）,神经嵴发育不全：由于神经嵴细胞（neuralcrestcells，NCC）发育缺陷或障碍而导致神经嵴出现异常的增殖、生存、迁徙和分化。由于NCC发育不良导致的这些组织和细胞的病变统称为神经嵴病。,研究背景,研究背景,研究背景,本研究Waardenburg综合征病例基因型和表型特征,研究背景,基因组水平：WS致病基因突变检测,后基因组水平：WS致病基因功能检测,分子水平：WS发病机制,临床水平：WS基因型如何导致表型,研究目的,前期研究,本文研究,表型基因型,基因型表型,？,研究内容,1,2,3,4,5,invitro,课程提纲,蛋白质加标签(Tag);定点突变；转染亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,蛋白质加上标签(Tag),Why：便于检测；市场上有很好的针对标签蛋白的抗体。What：Flag:DYKDDDDK；HA：YPYDVPDYA；c-Myc：EQKLISEEDL；V5：GKPIPNPLLGLDSTHow：前提是不能影响蛋白质的功能,课程提纲,蛋白质加标签(Tag);定点突变；转染亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,定点突变site-directedmutagenesis,QuikChangeSite-DirectedMutagenesisKit(Stratagene公司),突变引物设计,GoogleSearch“Quikchangeprimerdesign”,原始质粒,原始质粒改造,突变质粒构建,DNA测序鉴定,课程提纲,蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,转染-transfection,TransfectionisamethodoftransportingDNA,RNAand/orvariousmacromoleculesintoaneukaryoticcellbyusingchemical,lipidorphysicalbasedmethods.,Transfectionmethods,DEAEdextranCationicpolymerCalciumphosphate磷酸钙Electroporation电穿孔Microinjection显微注射Cationicliposome,DEAE-dextranDiethylaminoethyl(DEAE)-dextranisoneoftheoldestmethodsforintroducingnucleicacidsintoculturedmammaliancells.ThepositivelychargedDEAE-dextranmoleculeinteractswiththenegativelychargedphosphatebackboneofthenucleicacid.TheDNADEAEdextrancomplexesappeartoadsorbontothecellsurfaceandbetakenupbyendocytosis.优缺点：仅适合瞬转（transienttransfection），不适合稳转；细胞毒较高；重复性较好。,Cationicpolymer,Branched(A)andlinear(B)polyethyleneimine(PEI),商品名：ExGen500，Turbofect,CalciumPhosphate磷酸钙TheprincipleinvolvesmixingDNAinaphosphatebufferwithcalciumchloride.Theresultingcalcium-phosphateDNAcomplexesadheretothecellmembraneandenterthecytoplasmbyendocytosis.优缺点：适合稳转；重复性较差，主要影响因素包括溶液的pH值；一些细胞不适合磷酸钙转染。,CationicLiposome脂质体Theliposomescurrentlyinusetypicallycontainamixtureofcationicandneutrallipidsorganizedintolipidbilayerstructures.Transfection-complexformationisbasedontheinteractionofthepositivelychargedliposomewiththenegativelychargedphosphategroupsofthenucleicacid.TheuptakeoftheliposomeDNAcomplexesmaybemediatedbyendocytosis.品牌：Lipofectamine(Invitrogen)。优缺点：转染效率高；重复性好；转染时需降低血清浓度，从而提高了细胞毒性；,ElectroporationCellsinasuitablecuvettearesubjectedtoashorthigh-voltagepulsethatcausesthemembranepotentialofthecellstobreakdown.Asaresult,poresareformedthroughwhichmacromoleculessuchasDNAcanenter.优缺点：适合几乎所有的细胞；瞬转、稳转；需要DNA量大；高达5070%细胞死亡；,Microinjection,DirectMicroinjection:Useofafineneedle.HasbeenusedfortransferofDNAintoembryonicstemcells.,研究结果,野生/突变PAX3蛋白表达,野生/突变SOX10蛋白表达,A,B,课程提纲,蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,ReporterAssay:fortranscriptionalfactors(TF),Promoter,Geneofinterest,TF:transcriptionalfactorAD:activationdomainDBD:DNA-bindingdomain,ReporterGene:Agenewithareadilymeasurablephenotypethatcanbeeasilydistinguishedoverabackgroundofendogenousproteins.,Reportergenes,CAT：chloramphenicolacetyltransferase-gal：(-galactosidase)SEAP：(secretedalkalinephosphatase)LuciferaseGFP：GreenFluorescentProtein,CAT：chloramphenicolacetyltransferase氯霉素乙酰转移酶,1streportergeneusedtomonitortranscriptionalactivityincells;：Bacterialenzymethattransfersacetylgroupsfromacetyl-CoAtochloramphenicol,detoxifyingit;Reactionquantifiedusingradiolabeledsubstrates(14C-chloramphenicol)orbyELISA(non-radioactive).,CATassay：acetylated定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,亚细胞定位subcellularlocalization,免疫荧光（immunofluorescence）；细胞器分离。,Immunofluorescence,利用免疫荧光（immunofluorescence）技术染色目标蛋白；利用细胞器特异性抗体或者标记物染色细胞器；激光共聚焦显微镜观察，,Subcellularfraction,研究结果,1.野生/突变PAX3蛋白亚细胞定位,ZhangH,etal.HumGenet,2012,131:491-503.,研究结果,2.野生/突变SOX10蛋白亚细胞定位,ZhangH,etal.HumGenet,2012,131:491-503.,课程提纲,蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,DNA-蛋白质相互作用,ElectroMobilityShiftAssay(EMSA)DNAprecipitationChIP:Chromatinimmunoprecipitation,EMSA:Principle,R.Voll09/01,Adouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabeledwitharadioactiveisotopeandincubatedwithanuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.,NF-B,FreeProbe,RadioaktivelylabeledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-B,RadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe),Nuclearextractofnon-activatedcells,Nuclearextractofactivatedcells,R.Voll09/01,Supershift,Adouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabelledradioactiveandincubatedwithanuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.,p50/p65,Freeprobe,RadioaktivlabelledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-B,RadioactivlabelledoligonucleotidewithNF-B-bindingsite(probe),Nuclearextractofactivatedcells,Nuclearextractofactivatedcellswithanti-p50antibody,p50/p65+anti-p50,CompetitionwithUnlabeledOligos,IncreasingamountsofunlabeledoligoscontainingtheNF-kBbindingsiteorunlabeledoligoswithamutatedbindingsitewereaddedtothereactionmixpriortogelelectrophoresis.Specificbindingisextinguishedonlybythenon-mutatedoligo.,p50/p50,Freeprobe,p50/p65,WildtypeoligoMutatedoligo,GGGGACTTTCCC,GGAGACTTTCCC,BiotinylateddsDNA,Celllysate,Streptavidin-agrose,DetectproteinlevelwithWesternblot,MITFPAX3SOX10,ChIP：染色体共沉淀,课程提纲,蛋白质加标签(Tag);定点突变；转染;亚细胞定位；Reporterassay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。,Proteininteraction,Glutathione-S-Transferase(GST)pulldownYeastTwo-hybridCo-ImmunoprecipitationFluorescenceResonanceEnergyTransfer(FRET)SurfacePlasmonResonance(SPR),融合蛋白pull-down实验,融合蛋白pull-down技术基本原理是将一种蛋白质固定于某种基质上(如Sepharose),当细胞抽提液经过该基质时，可与该固定蛋白相互作用的配体蛋白被吸附，而没有被吸附的“杂质”则随洗脱液流出。被吸附的蛋白可以通过改变洗脱液或洗脱条件而回收下来。为了更有效地利用pull-down技术，可以将待纯化的蛋白以融合蛋白地形式表达，即将“诱饵”蛋白与一种易于纯化地配体蛋白相融合。1988年Smith等利用谷胱甘肽S转移酶（glutathione-S-transferase,GST）融合标签从细菌中一步纯化出GST融合蛋白。GST融合蛋白在经过固定有GSH(glutathione)的色谱柱时，就可以通过GST与GSH的相互作用而被吸附。当再有细胞抽提物过柱，就可得到能够与“诱饵”蛋白相互作用的兴趣蛋白。,免疫共沉淀,酵母双杂交,SurfacePlasmonResonance(SPR，表面等离子体共振),Oneinteractionpartner(Y)isboundtothemetalfilmwhiletheother(redballs)partnerIsinjectedoverthesurface.Amountofboundproteinisquantifiedbaseduponangleofreflectedlight.Realtimeassociationanddissociationofaprotein-proteininteractioncanbequantified.,SurfacePlasmonResonance(SPR),Frster/FluorescentResonanceEnergyTransfer(FRET),anon-radiative