科研设计ppt.ppt

细胞生物学研究思路的探讨,李家大 医学遗传学国家重点实验室 Email：,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）,神经嵴发育不全：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,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,in 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。,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：GKPIPNPLLGLDST How：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 mutagenesis,QuikChange Site-Directed Mutagenesis Kit (Stratagene 公司),突变引物设计,/CollectionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15,Google 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-transfection,Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods.,Transfection methods,DEAE dextran Cationic polymer Calcium phosphate 磷酸钙 Electroporation 电穿孔 Microinjection 显微注射 Cationic liposome,DEAE-dextran Diethylaminoethyl (DEAE)-dextran is one of the oldest methods for introducing nucleic acids 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； 细胞毒较高； 重复性较好。,Cationic polymer,Branched (A) and linear (B) polyethyleneimine (PEI),商品名： ExGen500， Turbofect,Calcium 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 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 liposome 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；,Electroporation Cells in a suitable cuvette are subjected to a short high-voltage pulse that causes the membrane potential of the cells to break down. As a result, pores are 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；,Microinjection,Direct Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells.,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； 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),Promoter,Gene of interest,TF: transcriptional factor AD: activation domain DBD: DNA-binding domain,Reporter Gene: A gene with a readily measurable phenotype that can be easily distinguished over a background of endogenous proteins.,Reporter genes,CAT ： chloramphenicol acetyltransferase -gal ： (-galactosidase) SEAP： (secreted alkaline phosphatase) Luciferase GFP： Green Fluorescent Protein ,CAT： chloramphenicol acetyltransferase 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： acetylated & non-acetylated chloremphenicol are cheched by TLC,CAT assay： ELISA,-gal (-galactosidase):,E. coli enzyme (encoded by lacZ) that hydrolyzes sugars such as lactose Many assay formats: colorimetric, fluorescent, chemiluminescent,SEAP (secreted alkaline phosphatase):,Secreted outside the cell (can assay 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) luciferase Sea pansy (Renilla reniformis) luciferase Firefly luciferase produces light by ATP-dependent,Photinus pyralis,Renilla reniformis,GFP: Green Fluorescent Protein,Derived from jellyfish Aequorea victoria Autofluorescent upon UV irradiation (doesnt require cofactors or substrates) Retains activity in presence of heat, denaturants, detergents, most proteases Allows 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,A,B,MITF promoter,MITF promoter,PAX3,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, 2012,131:491-503.,课程提纲,蛋白质加标签 (Tag); 定点突变； 转染; 亚细胞定位； 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 precipitation ChIP: Chromatin immunoprecipitation,EMSA: Principle,R. Voll 09/01,A double-stranded oligonucleotide containig a NF-B- binding site is labeled with a radioactive 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-B,Free Probe,Radioaktively labeled oligonucleotide with NF-B - binding site (probe) and bound NF-B,Radioactively labeled oligonucleotide with NF-B - binding site (probe),Nuclear extract of non-activated cells,Nuclear extract of activated cells,R. Voll 09/01,Supershift,A 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 oligonucleotide causes a shift compared to the free probe.,p50/p65,Free probe,Radioaktiv labelled oligonucleotide with NF-B - binding site (probe) and bound NF-B,Radioactiv labelled oligonucleotide with NF-B - binding site (probe),Nuclear extract of activated cells,Nuclear extract of activated cells with anti-p50 antibody,p50/p65 + anti-p50,Competition with Unlabeled Oligos,Increasing amounts of unlabeled oligos containing the NF-kB binding site or unlabeled oligos with a mutated binding site were added to the reaction mix prior to gel electrophoresis. Specific binding is extinguished only by the non-mutated oligo.,p50/p50,Free probe,p50/p65,Wild type oligo Mutated oligo,GGG GAC TTT CCC,GGA GAC TTT CCC,Biotinylated dsDNA,Cell lysate,Streptavidin-agrose,Detect protein level with Western blot,MITF PAX3 SOX10,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； Reporter assay； DNA-蛋白质相互作用； 蛋白质相互作用； 蛋白质半衰期。,Protein interaction,Glutathione-S-Transferase (GST) pulldown Yeast Two-hybrid Co-Immunoprecipitation Fluorescence 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”蛋白相互作用的兴趣蛋白。,洗脱 （2）,结合 （3）,检测 （6）,收集 （5）,洗脱 （4）,固定诱 饵蛋白 （1）,GST pulldown,免疫共沉淀,酵母双杂交,Surface Plasmon Resonance (SPR，表面等离子体共振),One interaction partner (Y) is bound to the metal film while the other (red balls) partner Is injected over the surface. Amount of bound protein is quantified based upon angle of reflected light. Real time association and dissociation of a protein-protein interaction can be quantified.,Surfac