1、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
2、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,
3、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: ATAACTTCGTATAATGTATGCTATACGAAGTTAT,顺式调控元件,（Deletion）,20,ATAACTTCGTATAATGTATGCTATACGAAGTTAT,ATAACTTCGTATAATGTATGCTATACGAAGTTAT,思考题如何研究转录因子A是否调控基因B？如何
4、调控基因B？,DNA 和蛋白质的相互作用EMSAChIP,R. Voll 09/01,Regulatory Region,Coding Sequence,Gene Regulation by Transcription Factors,Application:,R. Voll 09/01,Detection of DNA-binding factors/proteinsAnalysis of DNA sequences (e. g. promoter or enhancer regions) for their potential to bind specifically to protein
5、s/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 isotop
6、e 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 - bin
7、ding 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
8、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.
9、Centrifuge at high speed 8. Remove nuclear extract, determine protein concentrationand freeze on dry ice until EMSA is performed,The Probe,R. Voll 09/01,Double stranded radiolabeled oligonucleotides containing a transcription factor binding siteAP-1 5-GCT TGA TGA CTC AGC CGG AA C-3 3-CGA ACT ACT GAG
10、 TCG GCC TT G-5NF-kB 5-AGT TGA GGG GAC TTT CCC AGG C-3 3-TCA ACT CCC CTC AAA GGG TCC G-5Binding 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 Polynucleot
11、ide Kinase5-AGT TGA GGG GAC TTT CCC AGG-33-CA ACT CCC CTC AAA GGG TCC G-55-P-AGT TGA GGG GAC TTT CCC AGG-33-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,Co
12、mpetitor DNA: Competition of unspecific poly (dI-dC) . poly (dI-dC) binding (e. g. histones)Radiolabeled Probe: Detection of DNA-binding proteins Reaction Buffer Binding 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 Specifi
13、city,R. Voll 09/01,Supershift using antibodies against the DNA-binding proteinCompetition 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
14、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,Radioactive labelled oligonucleotide with NF-B - binding site (probe) and bound NF-B,Radioactiv labelled oligonucleotide with NF-B - binding site (probe
15、),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-kB 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,GGA GAC TTT CCC,
