《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()、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 kidneyNIH_ZGC_8:LiverNIH_ZGC_26:ovary,1 year old femaleNIH_ZGC_21:skin,normal adultNIH_ZGC_6:testisNIH_ZGC_27:whole body,30dpfNIH_ZGC_29:30dpf,whole body,probable malesSgano SJD adult male:whole body,adult maleNIH_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()、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:brainNIH_ZGC_15:embryo,2-8hpfNIH_ZGC_18:embryo,18-20hpfNCI_CGAP_Zemb2:embryo,24hpfGISZF001:embryo,0-72hpfNCI_CGAP_Zemb3:embryo,72hpfZebrafish adult retina cDNA:adult eye,retina 1-2 year oldNIH_ZGC_9:eye,72hpfNIH_ZGC_22:GillNIH_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-1Janus kinase-2Transforming growth factor-1Heat shock protein 70Heat shock protein 90Elongation factor-1Bleomycin hydrolaseCytochrome c oxidase subunit IICytochrome C oxidase subunit IIINovel 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 cattleMyostatin 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 ENUTreatment35 muatnt lines/320F2 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 mutationsAccessible to molecular analysisTag gene for expression analysisEfficient:single transfection gives 100s of mutationsBut,random insertions lack precision&intronic location,Identification of trapped genes by 5 RACE,AAAAAAAAAAAAAAAAAAAA,Exon 1,geo trap,RNA,AAAAAAAAAAAAAAAAAAAA,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 geo,Clone and sequence to identify Gene X,Gene X,geo primer 2,Whole Genome Mutagenesis:Advantages of gene traps:Pre-existing commercial collectionsTrap often creates hypomorphic or null allele3)Can use RT-PCR to screen for gene disruption,4)-Geo servers as histological or enzymatic assay of target gene expression,International Gene Trap Consortium,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,F030hpf,F15dpf,Screening for temperature-sensitive bacterial or yeast mutants,Screens can detect mutations that affect an animals 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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达产物的改变（组织细胞分布、时序特征、受刺激表达特性、基因表达的抑制）相互作用蛋白的寻找与核苷酸结合的活性(蛋白质对启动子调控的研究）改变表达特性导致的细胞表现型观察,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）,Results of a BLAST search,Sequence similarity can be provide clues about protein function,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（利用生物信息学分析可能的功能基团）,Domain information for zebrafish gasp,Protease inhibitor domain,DEAD domain:RNA bindingKRXXKRKR etc:nuclear localizatin signalZinc finger domain:DNA bindingHomeodomain:DNA binding:Secretary signal peptide analysis,Many domain analysis software on the web!,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)1。细胞、组织染色（Ab免疫染色）2。融合蛋白技术部（应用于蛋白定位、蛋白纯化提取）,Fusion proteins can be used to analyze protein function and to track proteins in living cells(1),Epitope tagging allows the localization or purification of proteins,Fusion proteins can be used to analyze protein function and to track proteins in living cells(2),Fluorescence resonance energy transfer(FRET).,Three types of matrices used for chromatography,Purification of protein complexes using a GST-tagged fusion protein,Popular Epitope tagging for fusion protein studies:,GST(glutathione S-transferase,25kDa)FLAG(“DYKDDDDK”,synthesized)HIS(“HHHHHH”,synthesized)C-MYC(“EQKLISEEDL”,part of c-myc,synthesized)HA(“YPYDVPDYA”,part of hemagglatin,synthesized)VSV-G(“YTDIEMNRLGK”,synthesized)HSV(“QPELAPEDPED”,synthesized)V5(“GKPIPNPLLGLDST”,synthesized)GFP(about 235aa,47kDa),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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达产物的改变（组织细胞分布、时序特征、受刺激表达特性、基因表达的抑制)1）基因过量表达（强启动子调控野生型或突变型蛋白的细胞转染实验）,蛋白结合功能区,蛋白调控功能区,pSV40，or pCMV,or other specific promoter,蛋白结合功能区,突变的蛋白调控功能区,pSV40，or pCMV,or other specific promoter,Dominant negative form,Wild-type form,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达方式的改变（组织细胞分布、时序特征、受刺激表达特性、基因表达的抑制)1）基因过量表达（强启动子调控野生型或突变型蛋白的细胞转染实验）2）特定外界刺激反应条件（激素或细胞因子、营养条件、分化状况、物理化学刺激等）,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达产物的改变（组织细胞分布、时序特征、受刺激表达特性、基因表达的抑制)1）基因过量表达（强启动子调控野生型或突变型蛋白的细胞转染实验）2）特定外界刺激反应条件（激素或细胞因子、营养条件、分化状况、物理化学刺激等）3）基因的mRNA的降解破坏,RNAi Introduction,RNAi=RNA interferenceThe term used to describe the interference of RNA as a natural mechanism,and also as a scientific research tool.,The silencing of gene expression by double-stranded RNA molecules.,RNAi Introduction,Naturally RNAi acts as a gene defence mechanism.Its purpose is to silence types of RNA that are not normally produced by cells such as RNA viruses and rogue genetic elements.RNAi is a useful tool to find out the function of a gene.,RNAi Introduction,Early days of molecular biology:Forward GeneticsAnalysis that starts with phenotype to determine the genotype.Trait Identification DNA Sequencing Mutation Identification Gene FunctionModern times:Reverse GeneticsControlled manipulation of genes to determine phenotype.DNA Sequencing Gene manipulation Gene Function,(siRNA),siRNA Introduction,siRNA are short dsRNA molecules 21 nucleotide strands of RNA in a staggered duplex-19 nucleotides double-stranded,with 2 base overhangs.,siRNA is created when an enzyme known as Dicer cleaves dsRNA into the short duplexed structure known as siRNA.,siRNA Introduction,siRNA Introduction,Mechanism for RNAi to knock down RNA product,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达方式的改变（组织细胞分布、时序特征、受刺激表达特性）相互作用蛋白的寻找与核苷酸结合的活性(蛋白质对启动子调控的研究）改变表达特性导致的细胞表现型观察,From Current Protocols,Co-Immuno-precipitation(Co-IP),From Current Protocols,GST pulldown,Cellular localization of the C114 protein(1),Yin Z.,et al.(2003).JBC.278:22838-22845.,Cellular localization of the C114 protein(2),Yin Z.,et al.(2003).JBC.278:22838-22845.,Nucleotide binding analysis of the C114 protein,Yin Z.,et al.(2003).JBC.278:22838-22845.,C114 interacts with PKR(1),Yin Z.,et al.(2003).JBC.278:22838-22845.,C114 interacts with PKR(2),Yin Z.,et al.(2003).JBC.278:22838-22845.,C114 interacts with PKR(3),Yin Z.,et al.(2003).JBC.278:22838-22845.,Association of C114 and PKR inhibits PKR activation(1),Yin Z.,et al.(2003).JBC.278:22838-22845.,Association of C114 and PKR inhibits PKR activation(2),Yin Z.,et al.(2003).JBC.278:22838-22845.,Association of C114 and PKR inhibits PKR activation(3),Yin Z.,et al.(2003).JBC.278:22838-22845.,IgGbeads,Protein ofinterest,CBP,ProtA,TEVsite,Boundproteins,Wash outcontaminants,TEVprotease,Calmodulinbeads,EGTA,Tandem Affinity Purification(TAP),After Rigaut et al.(1999)Nature Biotech.17:1030,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达方式的改变（组织细胞分布、时序特征、受刺激表达特性）相互作用蛋白的寻找与核苷酸结合的活性(蛋白质对启动子调控的研究）改变表达特性导致的细胞表现型观察,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（利用生物信息学分析可能的功能基团）基因编码蛋白的细胞内定位（胞内转位现象，trans-localization)基因表达方式的改变（组织细胞分布、时序特征、受刺激表达特性）相互作用蛋白的寻找与核苷酸结合的活性(蛋白质对启动子调控的研究）改变表达特性导致的细胞表现型观察,The modular structure of a gene activator protein,The yeast two-hybrid system for detecting protein-protein interactions,Invitrogen,Invitrogen,Two-hybrid prey vector,B42=transcription activation domain,DUALmembrane 2-hybrid system(DUALsystems),Bait,Prey,Split-ubiquitin,A map of the protein protein interactions observed between different functional groups of proteins in the yeast S.cerevisiae,