顺式调控元件的寻找20121116

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,*,*,*,分子生物学工作基础,基因的表达调控,刘喆,3,思考题：,如何研究顺式调控元件的功能？,4,Luciferase,Assay,基因敲除,5,Luciferase,Assay,6,Luciferase,Assay,质粒构建,细胞转染,Dual,Luciferase,分析,24-48,小时,7,细胞转染,电转染,磷酸钙转染,脂质体转染,（,lipofectamine,2000,）,8,电转染,细胞上短时间暂时性的穿孔让外源质粒进入,9,磷酸钙转染,通过,DNA,，氯化钙与磷酸缓冲液混合，沉淀形成保护,DNA,且极小不溶的磷酸钙颗粒。这些磷酸钙,-DNA,复合物粘附到细胞膜并通过胞饮作用进入细胞中,10,脂质体转染,膜融合的方法将外源,DNA,导入细胞,11,转染效率的矫正,共转染：,Firfly,和,Renilla,萤火虫,(,Photinus,pyralis,),萤光素酶和海肾,(,Renilla,reniformis,),萤光素酶,Promoter and,cisregulatory,elements,exon1,Cisregulatory,element,enhancer,silencer,insulator,CG island,MAR,DNA tethering fragment,exon1,Cisregulatory,element,Luciferase,P,Cisregulatory,element,14,15,16,17,Luciferase,Assay,的功能,确定启动子位置,判断顺式调控元件的功能,检测转录因子是否能够直接调控基因的表达,初步判断基因表达变化的原因,18,基因条件性敲除,确定启动子位置,判断顺式调控元件的功能,检测转录因子是否能够直接调控基因的表达,初步判断基因表达变化的原因,19,LoxP,:,ATAACTTCGTATA,ATGTATGC,TATACGAAGTTAT,顺式调控元件,（,Deletion,）,20,ATAACTTCGTATA,ATGTATGC,TATACGAAGTTAT,ATAACTTCGTATA,ATGTATGC,TATACGAAGTTAT,思考题,如何研究转录因子,A,是否调控基因,B,？如何调控基因,B,？,DNA,和蛋白质的相互作用,EMSA,ChIP,R.,Voll,09/01,Regulatory Region,Coding Sequence,Gene Regulation by Transcription Factors,Application:,R.,Voll,09/01,Detection of DNA-binding factors/proteins,Analysis of DNA sequences(e.g.promoter or enhancer regions)for their potential to bind specifically to proteins/nuclear extracts,Analysis of(sub-)cellular extracts for the presence of certain DNA-binding proteins(e.g.a transcription factor with a known recognition sequence),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,Preparation of Nuclear and,Cytosolic,Extracts,R.,Voll,09/01,The procedure is carried out on ice,rsp,at 4C and in the,presence of protease(and,phosphatase,)inhibitors.,1.Swell cells in hypotonic,lysis,buffer,2.Add NP-40 and vortex to disrupt,cytoplasmic,membrane,3.Centrifuge to pellet nuclei,4.Carefully remove supernatant(contains,cytosolic,and,membrane fraction),4.Wash nuclear pellet once in,lysis,buffer,5.Add hypertonic extraction buffer to nuclear pellet,6.Agitate,vigouresly,for 30 minutes,7.Centrifuge at high speed,8.Remove nuclear extract,determine protein concentration,and freeze on dry ice until EMSA is performed,The Probe,R.,Voll,09/01,Double stranded,radiolabeled,oligonucleotides,containing a transcription factor binding site,AP-1,5-GCT TGA,TGA,CTC AGC CGG AA C-3,3-CGA ACT,ACT,GAG TCG GCC TT G-5,NF-,k,B,5-AGT TGA GGG GAC TTT CCC AGG C-3,3-TCA ACT CCC CTC AAA GGG TCC G-5,Binding motif,R.,Voll,09/01,Annealing the,Oligos,Heat up an,equimolar,mixture of the 2,oligos,to 95C and let them slowly cool down by turning,off the heat block.,Labeling the Probe,R.,Voll,09/01,A.T4 Polynucleotide,Kinase,5-AGT TGA GGG GAC TTT CCC AGG-3,3-CA ACT CCC CTC AAA GGG TCC G-5,5-P-AGT TGA GGG GAC TTT CCC AGG-3,3-CA ACT CCC CTC AAA GGG TCC G-P-5,PNK,+,Adenosin,-P-P-P(,g,-ATP),Removal of free radioactive material,R.,Voll,09/01,Remove not incorporated,isotop,by,Sephadex,G50 column,Reagents,R.,Voll,09/01,Competitor DNA:Competition of unspecific,poly(,dI-dC,),.,poly(,dI-dC,)binding(e.g.,histones,),Radiolabeled,Probe:Detection of DNA-binding,proteins,Reaction BufferBinding conditions,Analysis by non-Denaturing,Polyacrylamide,Gel Electrophoresis,R.,Voll,09/01,准备非变性,PAGE,凝胶,上样,电泳,干胶,放射自显影,Proof of Specificity,R.,Voll,09/01,Supershift,using antibodies against the DNA-binding protein,Competition for binding to the,radiolabeled,probe using,unlabeled,wildtype,and mutated,oligos,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,Radioa,c,tiv,e,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,R.,Voll,09/01,Competition with Unlabeled,Oligos,Increasing amounts of unlabeled oligos containing the,NF-,k,B 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,Unspecific,Wild type,oligo,Mutated,oligo,GGG GAC TTT CCC,GG,A,GAC TTT CCC,