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1、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
2、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：GKPIPN
3、PLLGLDST 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,https:/ 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;
4、 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 E
5、lectroporation 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 th
6、e 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 princip
7、le 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
8、 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
9、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 a
10、s 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
11、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,C
12、AT ： 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
13、 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
14、 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 measu
15、ring 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,Re
16、nilla 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
