《基因研究方法》ppt课件.ppt

分子细胞生物学研究方法,(一)功能基因的筛选方法,特异性功能基因的筛选和寻找方法: 1）对表达于特定组织、特定阶段的功能基因的筛选 2）对在特定的生理过程中表达的的功能基因的筛选 3）自然突变及基因诱变的表现型筛选观察与其基因定位 4）同已知的具有特定功能的基因的相关基因的筛选和研究,,对表达于特定组织、特定阶段的功能基因的筛选,一、文献查阅,,对表达于特定组织、特定阶段的功能基因的筛选,一、文献查阅 二、充分利用GeneBank、SymAtlas(http://symatlas.gnf.org/SymAtlas/)、 ZFIN等网上公共资源 a. 直接查阅基因表达信息,Mouse jumonji,Human RAN binding protein 1,NCI_CGAP_Zkid1: adult kidney NIH_ZGC_8: Liver NIH_ZGC_26: ovary, 1 year old female NIH_ZGC_21: skin, normal adult NIH_ZGC_6: testis NIH_ZGC_27: whole body, 30dpf NIH_ZGC_29: 30dpf, whole body, probable males Sgano SJD adult male: whole body, adult male NIH_ZGC_28: whole body, female,De Ruijter AJ et al. 2003. Biochem. J. 370: 737-749. (review),HDAC expression in various normal and cancerous cells,,E11.5,E14.5,HDAC2 highly expresses in heart region during embryonic development,E12.5 15.5 17.5 P 3 15 25 65,,-HDAC2,-tubulin,HDAC2 expression levels in heart,Yin Z et al. (2007). Nat. Med. 13, 324-331.,,对表达于特定组织、特定阶段的功能基因的筛选,一、文献查阅 二、充分利用GeneBank、SymAtlas(http://symatlas.gnf.org/SymAtlas/)、 ZFIN等网上公共资源 a. 直接查阅基因表达信息 b. 对大量的est资源进行利用和分析: 如熟知斑马鱼的一些被大量阅码的常用cDNA文库： NIH_ZGC_4: brain NIH_ZGC_15: embryo, 2-8hpf NIH_ZGC_18: embryo, 18-20hpf NCI_CGAP_Zemb2: embryo, 24hpf GISZF001: embryo, 0-72hpf NCI_CGAP_Zemb3: embryo, 72hpf Zebrafish adult retina cDNA: adult eye, retina 1-2 year old NIH_ZGC_9: eye, 72hpf NIH_ZGC_22: Gill NIH_ZGC_17: adult heart,鱼类研究的遗传资源,Cossins A and Crawford DL. 2005. Nat. Rev. in Genetics, 6, 324-333.,一、文献查阅 二、充分利用网上公共资源 三、对组织细胞特定过程中差异表达基因的筛选,对表达于特定组织、特定阶段的功能基因的筛选,Mehta D. et al., 2010. Curr. Psychiatry Rep. 12, 135-144.,基因表达的研究方法,Yin Z, Kwang J & Sin YM. Carp interleukin-1 beta in the role of an immuno-adjuvant FISH & SHELLFISH IMMUNOLOGY 10 (4): 375-378 MAY 2000,,Con A treatment,control,,,Driver cDNA,Test cDNA,cDNA subtraction cloning,Induced cDNA List: Interleukin-1 Janus kinase-2 Transforming growth factor-ß1 Heat shock protein 70 Heat shock protein 90 Elongation factor-1ß Bleomycin hydrolase Cytochrome c oxidase subunit II Cytochrome C oxidase subunit III Novel ests,Percoll,Yin Z., et al. (2003). JBC. 278: 22838-22845.,Using DNA microarrays to monitor the expression of thousands of genes simultaneously,Using cluster analysis to identify sets of genes that are coordinately regulated,,HDAC2 transcripts were 45 fold down regulated in 2 sets of HDAC2-/- ventricle samples,WT1,WT2,null2,null1,TABLE 2. Comparison of some gene expression levels in HDAC2 null heart with microarray analysis,,Identified Pathway (DAVIS): NFAT and Hypertrophy of the heart (Transcription in the broken heart)(Mus musculus) suppressed in HDAC2 null,,,,,,,,,,Myocyte enhancer factor 2C (MEF2c),Gata binding protein 4 (Gata 4),Thymoma viral proto-oncogene 1 (Akt),Calmodulin 1,Alpha-actin, skeleton muscle (-actin),Cossins A and Crawford DL. 2005. Nat. Rev. in Genetics, 6, 324-333.,特异性功能基因的筛选和寻找方法: 1）对表达于特定组织、特定阶段的功能基因的筛选 2）对在特定的生理过程中表达的的功能基因的筛选 3）自然突变及基因诱变的表现型筛选观察与其基因定位 4）同已知的具有特定功能的基因的相关基因的筛选和研究,对基因突变的个体的获取及分析,一、对自然突变的观察、收集和研究,Double muscle cattle Myostatin mutant,Splotch mice,对基因突变的个体的获取及分析,一、对自然突变的观察、收集和研究 二、基因诱变导致突变品种的产生,对基因突变的个体的获取及分析,一、对自然突变的观察、收集和研究 二、基因诱变导致突变品种的产生 1。

物理诱变 （如放射线、紫外线照射） 2化学诱变剂 （如ENU， N-ethyl-N-nitrosourea),Generation of mutants with developmental defects in zebrafish by ENU mutagenesis,Peng J. et al. (2004). Chinese Sci. Bullet. 49: 2154-2158.,3.0 mM ENU Treatment 35 muatnt lines/320 F2 families,对基因突变的个体的获取及分析,一、对自然突变的观察、收集和研究 二、基因诱变导致突变品种的产生 1物理诱变 （如放射线、紫外线照射） 2化学诱变剂 （如ENU， N-ethyl-N-nitrosourea) DNA插入 a. DNA片段插入 b. 病毒插入 c. 转座子插入,,,Gene trapping,bgeo reporter,SA,,ATG,,pA,,,,,,,Insertional mutations Accessible to molecular analysis Tag gene for expression analysis Efficient: single transfection gives 100s of mutations But, random insertions lack precision & intronic location,,,,Identification of trapped genes by 5’ RACE,,,,AAAAAAAAAAAAAAAAAAAA,,,Exon 1,ß geo trap,RNA,,AAAAAAAAAAAAAAAAAAAA,,,ß geo primer 1,Prime cDNA with Reverse Transcriptase,5’,3’,Enzymatically add tag sequence to cDNA 3’ end,cDNA,5’,3’,,,3’,,,PCR amplify with primers to tag sequence and ßgeo,Clone and sequence to identify Gene X,,Gene X,ß geo primer 2,Whole Genome Mutagenesis: Advantages of gene traps: Pre-existing commercial collections Trap often creates hypomorphic or null allele 3) Can use RT-PCR to screen for gene disruption,4) ß-Geo servers as histological or enzymatic assay of target gene expression,International Gene Trap Consortium,http://www.igtc.org.uk,Mutations available for 36% of genes,Tol2转座子介导的基因增效基因诱捕技术,Kawakami K. 2005. Dev. Dyn. 234: 244-254. Parinov S. et al. 2004. Dev. Dyn. 231: 449-459.,建立Tol2转座子增效基因诱捕的转基因斑马鱼诱变品系库,突变品系的筛选工作：已注射近400粒斑马鱼受精卵，目前筛选成功率为2/20, 尚有近200尾有待筛选检查,F0 30hpf,F1 5dpf,Screening for temperature-sensitive bacterial or yeast mutants,Screens can detect mutations that affect an animal's behavior,特异性功能基因的筛选和寻找方法: 1）对表达于特定组织、特定阶段的功能基因的筛选 2）对在特定的生理过程中表达的的功能基因的筛选 3）自然突变及基因诱变的表现型筛选观察与其基因定位 4）同已知的具有特定功能的基因的相关基因的筛选和研究,基因功能的体外和体内分析方法 一、体外方法： 文献查阅 基因序列分析-1（同源基因的寻找和功能比较） 基因序列分析-2 。