基因表达调控课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,基因表达与调控,基因表达与调控,1,研究基因转录调控的方法,Reporter gene assay,EMSA(Electrophoretic mobility shift assay),Foot-pringting,Methylation assay,Tet/off and Tet/on assay,Northern Blotting,ChIP,SAGE,Western blotting assay,研究基因转录调控的方法,2,Chromatin Immunoprecipitation(CHIP),-real-time analysis for protein/DNA interaction,You must know or have;,1.DNA sequence around interesting bind sites,2.Proteins that could bind to the sites,3.Antibody for the protein,Chromatin Immunoprecip,3,Example:RNA Pol II at transcription start point,-Nucleic Acids Research,2004,32(11):1-8,Obtain tissues from animal or human,Crosslink chromatin and binding proteins,Treat tissues by 1%formaldehyde for 12 min and then stop reaction by 0.125M Glycine,Disaggregate tissue by homogenizer,collect cells,Break cells by buffer containing 0.5%NP-40,spin to collect nuclei,Break nuclei by buffer containing 1%SDS,Sonicate chromatin into 500bp fragments,Block protein A/G agarose by,l,DNA,tRNA and BSA,Example:RNA Pol II at tr,4,Incubate sonicated DNA with blocked proteinA/G agarose to remove non-specific bind,spin to get supernatant,save sample as“Input”,Incubate pre-cleaned supernatant with anti-RNA Pol II antibody,Add blocked protein A/G agarose into reaction,Spin to collect protein A/G agarose and wash,Incubate sonicated DNA with bl,5,Elute chromatin DNA by 1%SDS,100mM NaHSO3,Incubate to reverse crosslink and use the,sample as PCR temperate,Design PCR primers that flack the,binding site(200500bp),15.Rea-time PCR,Elute chromatin DNA by 1%SDS,6,基因表达调控课件,7,S,erial,A,nalysis of,G,ene,E,xpression,(SAGE),-分析特定细胞或组织中的基因表达谱,Serial Analysis of Gene E,8,SAGE is based on the following principle:,9 bp DNA tag contains sufficient information to uniquely identify a mRNA,NNNN 4,4,=256,NNNNN 4,5,=1024,NNNNNN 4,6,=4096,NNNNNNN 4,7,=16376,NNNNNNNN 4,8,=65504,NNNNNNNNN 4,9,=262016,基因表达调控课件,9,BsmF1:,GGGACNNNNNNNN,NNNNNNNNNNNN,CCCTGNNNNNNNNNNNN,NNNNNNNN,NlaIII:NNNNCATGNNNNN,NNNNGTACNNNNN,Streptavidin:,A protein produced by the bacterium Streptomyces avidinii.It has four binding sites for biotin.It has been used extensively as probes in immunochemical systems,conjugated to antibodies,enzymes or fluorochromes.,BsmF1:,10,Synthesize double strand cDNA from mRNA used biotinylated Oligo dT primer,2.Cut cDNA by Nla III and most 3-end portion was isolated by binding to streptavidin beads,3.divide cDNA in half (A and B),Synthesize double strand cDNA,11,4.Ligate adaptor A or B to each half of cDNA,4.Ligate adaptor A or B to ea,12,5.Cut DNA by BsmF1 and blunt ends T4 DNA polymerase,6.Ligate A and B and amplify ditag by PCR with adaptor spercific,primers,5.Cut DNA by BsmF1 and blunt,13,7.Cut PCR product by Nla III and purify di-tag,8.Ligate to concatenation,9.Clone into Sph1 site in pSL301,NNNNNNGCATGCNNNNNNNN,NNNNNNCGTACGNNNNNNNN,10.DNA sequencing,7.Cut PCR product by Nla III,14,基因表达调控课件,15,Bert Vogelstein,Bert Vo,16,Tet-Off/Tet-On,Tetracycline(Doxycycine)controlled gene expression system,Te,17,Background:,Gene Structure of Tn10 Operon,TetR:repressor for tetA,can bind with Tetracycline,T:Transposase critical for operon jumping,Re:Resolvase to relaxation of twists in DNA helix,TetA,:,Actively expel intracellular tetracycline out of the cell,18,TRE:,Tetracycline-response element,in the promoter of tetA,5-TCCCTATCAGTGATAGAGA-3,bind with TetR,tTA:,Tetracycline-controlled Trans-Activator,a recombinant protein by,TetR(1-207Aa)VP16 C-terminal activating domain(127Aa),rtTA:,Base on the reverse TetR(rTetR)which has 4 amino acid mutates compares with wild type TetR,rTetR(1-207Aa)VP16 C-terminal activating domain(127Aa),TRE:,19,TRE in retrovirus plasmid pRev/TRE,7 X TRE,Response,virus,TRE in retrovirus plasmid pRev,20,tTA in pRevTet-off rtTA in pRevTet-on,Regulation virus,tTA in pRevTet-off,21,Tetracycline,Doxycycline,Tetracycline Doxycycline,22,Tetracycline,Doxycycline,Tetracycline Doxycycline,23,基因表达调控课件,24,Tet-off/Tet-on protocols,Make response retrovirus pRevTRE-X,Clone your gene of interest into pRevTRE,Target gene,25,Tet-off/Tet-on protocols,Transfect pRevTRE-X,pRevTet-off/pRevTet-on into packaging cells(PT67)separately,Response virus and regulate virus lack viral gene gag,pol and env that can be supplied by packaging cells,PT67 Package Cells,3 different viral particles,26,Tet-off/Tet-on protocols,Collect viral particles from culture medium,If Tet-off:,Infect target cells by:pRevTet-off viral+pRevTRE-X viral;,Select cells by G418/Hyg;,Culture cells in the medium with doxycycline;,Before experiment,remove doxycycline.,27,Tet-off/Tet-on protocols,5.If Tet-on:,Infect target cells by:pRevTet-on viral+pRevTRE-X viral;,Select cells by G418/Hyg;,Culture cells in the medium without doxycycline;,Before experiment,add doxycycline.,28,Determining expression of target gene:,Assay for mRNA -PCR,Northern blotting,for Protein-enzyme activity,Western blotting,7.Determining effects of expression of interest gene on whatever you want to study.,Determining expression of targ,29,