细胞生物学的研究思路详解演示文稿课件

1、细胞生物学的研究思路详解演示文稿（优选）细胞生物学的研究思路研究背景Waardenburg综合征（Waardenburg syndrome，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）分型致病基因临床表型特征WS1PAX3耳聋、着色异常、眼距增宽WS2MITF、SOX10、SNAI2耳聋、着色异常WS3PAX3耳聋、着色异常、上肢发育异常(Klein-Waardenburg 综合征)WS4SOX10、END3、ENDBR耳聋、着色异常、巨结肠(Shah-Waardenburg 综合征)神经嵴发育不全：

2、由于神经嵴细胞（neural crest cells，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。研究背景研究背景分型致病基因氨基酸改变耳聋虹膜异色皮肤色素改变内眦异位巨结肠WS2MITFR217I先天性亮蓝色虹膜面部大量雀斑WS2MITFT192fs先天性完全性虹膜异色WS1PAX3H80D先天性完全性虹膜异色+WS1PAX3H186fs先天性亮蓝色虹膜+WS2SOX10E248fs先天性亮蓝色虹膜WS2SOX10G37fs先天性亮蓝色虹膜WS2SOX10G38fs先天性亮蓝色虹膜WS2SOX10R43X先天

3、性完全性虹膜异色面部少量雀斑WS4SOX10W85X先天性完全性虹膜异色+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？研究内容蛋白表达水平PAX3与SOX10相互作用活性检测蛋白亚细胞定位蛋白与DNA的结合MITFPAX3SOX1012345in vitro 课程提纲 蛋白质加标签 (Tag);定点突变；转染亚细胞定位；Reporter assay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期

4、。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；Reporter assay；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-directed mutagenesisQuikChange Site-Directed Mutagenesis Kit (Stratagene 公司)突变引物设计/Collec

5、tionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15Google Search “Quikchange primer design” 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；Reporter assay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。转染-transfectionTransfection is a method of transporting DNA, RNA and/or various macromolecules in

6、to an eukaryotic cell by using chemical, lipid or physical based methods.Transfection methods DEAE dextranCationic polymerCalcium phosphate 磷酸钙Electroporation 电穿孔Microinjection 显微注射Cationic liposomeDEAE-dextran Diethylaminoethyl (DEAE)-dextran is one of the oldest methods for introducing nucleic aci

7、ds into cultured mammalian cells. The positively charged DEAE-dextran molecule interacts with the negatively charged phosphate backbone of the nucleic acid. The DNADEAE dextran complexes appear to adsorb onto the cell surface and be taken up by endocytosis. 优缺点：仅适合瞬转 （transient transfection），不适合稳转；细

8、胞毒较高；重复性较好。Cationic polymerBranched (A) and linear (B) polyethyleneimine (PEI) 商品名： ExGen500， TurbofectCalcium Phosphate磷酸钙 The principle involves mixing DNA in a phosphate buffer with calcium chloride. The resulting calcium-phosphateDNA complexes adhere to the cell membrane and enter the cytoplasm

9、by endocytosis. 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。Cationic Liposome 脂质体The liposomes currently in use typically contain a mixture of cationic and neutral lipids organized into lipid bilayer structures. Transfection-complex formation is based on the interaction of the positively charged lipo

10、some with the negatively charged phosphate groups of the nucleic acid. The uptake of the liposomeDNA complexes may be mediated by endocytosis.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；ElectroporationCells in a suitable cuvette are subjected to a short high-voltage pulse that

11、causes the membrane potential of the cells to break down. As a result, poresare formed through which macromolecules such as DNA can enter.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；MicroinjectionDirect Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells.研究结

12、果野生/突变PAX3蛋白表达野生/突变SOX10蛋白表达AB 课程提纲 蛋白质加标签 (Tag);定点突变；转染;亚细胞定位；Reporter assay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。Reporter Assay: for transcriptional factors (TF)PromoterGene of interestTFDBDADPromoterReporter geneTFDBDADTF: transcriptional factorAD: activation domainDBD: DNA-binding domainReporter Gene: A g

13、ene with a readily measurable phenotype that can be easily distinguished over a background of endogenous proteins. Reporter genesCAT ： chloramphenicol acetyltransferase-gal ： (-galactosidase)SEAP： (secreted alkaline phosphatase)Luciferase GFP： Green Fluorescent Protein CAT： chloramphenicol acetyltra

14、nsferase氯霉素乙酰转移酶1st reporter gene used to monitor transcriptional activity in cells;：Bacterial enzyme that transfers acetyl groups from acetyl-CoA to chloramphenicol, detoxifying it;Reaction quantified using radiolabeled substrates (14C-chloramphenicol) or by ELISA (non-radioactive).CAT assay： acety

15、lated & non-acetylated chloremphenicol are cheched by TLCCAT assay： ELISA-gal (-galactosidase):E. coli enzyme (encoded by lacZ) that hydrolyzes sugars such as lactoseMany assay formats: colorimetric, fluorescent, chemiluminescentSEAP (secreted alkaline phosphatase): Secreted outside the cell (can as

16、say sample repeatedly and non-destructively by sampling culture medium) This protein is quantified directly by measuring the enzyme activity in the supernatant of the culture medium. Fluorescence and chemiluminescence assays are available for detection.Luciferase:Firefly (Photinus pyralis) luciferas

17、eSea pansy (Renilla reniformis) luciferaseFirefly luciferase produces light by ATP-dependent Photinus pyralis Renilla reniformisGFP: Green Fluorescent ProteinDerived from jellyfish Aequorea victoriaAutofluorescent upon UV irradiation (doesnt require cofactors or substrates)Retains activity in presen

18、ce of heat, denaturants, detergents, most proteasesAllows for non-invasive monitoring of gene expression in living tissues研究方法荧光素酶活性检测（Luciferase activity assay）研究结果ABMITF promoterMITF promoterPAX3 SOX10 研究结果E248fs突变对野生SOX10蛋白产生显性负效应作用 课程提纲 蛋白质加标签 (Tag);定点突变；转染;亚细胞定位；Reporter assay；DNA-蛋白质相互作用；蛋白质相互

19、作用；蛋白质半衰期。亚细胞定位 subcellular localization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，Subcellular fraction研究结果1.野生/突变PAX3蛋白亚细胞定位Zhang H, et al. Hum Genet, 2012,131:491-503. 研究结果2.野生/突变SOX10蛋白亚细胞定位Zhang H, et al. Hum Genet, 2012,13

20、1:491-503. 课程提纲 蛋白质加标签 (Tag);定点突变；转染;亚细胞定位；Reporter assay；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Electro Mobility Shift Assay (EMSA)DNA precipitationChIP: Chromatin immunoprecipitationEMSA: PrincipleR. Voll 09/01A double-stranded oligonucleotide containig a NF-B- binding site is labeled with a radioa

21、ctive isotope and incubated with a nuclear extract.During gel-electrophoresis, NF-B bound to the oligonucleotide causes a shift compared to the free probe.NF-BFree ProbeRadioaktively labeled oligonucleotidewith NF-B - binding site (probe) and bound NF-B Radioactively labeled oligonucleotide with NF-

22、B - binding site (probe)Nuclear extract of non-activated cellsNuclear extract ofactivated cellsR. Voll 09/01SupershiftA double-stranded oligonucleotide containig a NF-B- binding site is labelled radioactive and incubated with a nuclear extract.During gel-electrophoresis, NF-B bound to the oligonucle

23、otide causes a shift compared to the free probe.p50/p65Free probeRadioaktiv labelled oligonucleotidewith NF-B - binding site (probe) and bound NF-B Radioactiv labelled oligonucleotide with NF-B - binding site (probe)Nuclear extract of activated cellsNuclear extract of activated cells with anti-p50 a

24、ntibodyp50/p65 + anti-p50Competition with Unlabeled OligosIncreasing amounts of unlabeled oligos containing the NF-kB binding siteor unlabeled oligos with a mutated binding site were added to the reaction mixprior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo

25、.p50/p50Free probep50/p65Wild type oligo Mutated oligoGGG GAC TTT CCCGGA GAC TTT CCCBBBiotinylated dsDNACell lysateStreptavidin-agroseBDetect protein level with Western blotMITF PAX3 SOX10 ChIP：染色体共沉淀Negative controlAmplify DNA and LabelCross-link whole cells with formaldehydeIsolate genomic DNAAdd

26、protein-specific antibodyImmunoprecipitate and purify imminocomplexesReverse cross-links and purify DNASonicate DNA to produce sheared, soluble chromtinChIP- PCRTarget geneNo DNAInput DNAIgGSp1c-Fosc-JunPCRHybridize to arraysChIP- chipsequencingRegionsequencedChIP- seq 课程提纲 蛋白质加标签 (Tag);定点突变；转染;亚细胞定

27、位；Reporter assay；DNA-蛋白质相互作用；蛋白质相互作用；蛋白质半衰期。Protein interactionGlutathione-S-Transferase (GST) pulldownYeast Two-hybridCo-ImmunoprecipitationFluorescence Resonance Energy Transfer (FRET)Surface Plasmon Resonance (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