基因表达调控3.ppt

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1、基因表达与调控,邱赫殷毖妆谎董押颜罩瑟摆溶畴赴色捉察质狂纪淮饵唬卑今仕鞠艳斩隙碱基因表达调控-3基因表达调控-3,研究基因转录调控的方法Reporter gene assay EMSA(Electrophoretic mobility shift assay)Foot-pringtingMethylation assay Tet/off and Tet/on assayNorthern Blotting ChIPSAGEWestern blotting assay,挪硼菠计定函徐凛涧韦须乱罩志茂敞短数潦筑淫酥返摔附鱼潞扮廷韵楚英基因表达调控-3基因表达调控-3,Chromatin Immuno

2、precipitation(CHIP)-real-time analysis for protein/DNA interactionYou must know or have;1.DNA sequence around interesting bind sites 2.Proteins that could bind to the sites 3.Antibody for the protein,安卓堤个搓敷呆藉喻闯幻若脉喝盘择编啤估隅滁轻抄署吮劝鱼蛙篇臃鸽巨基因表达调控-3基因表达调控-3,Example:RNA Pol II at transcription start point-Nuc

3、leic Acids Research,2004,32(11):1-8Obtain 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 GlycineDisaggregate tissue by homogenizer,collect cellsBreak cells by buffer containing 0.5%NP-40,spin to collec

4、t nucleiBreak nuclei by buffer containing 1%SDSSonicate chromatin into 500bp fragmentsBlock protein A/G agarose by lDNA,tRNA and BSA,铱焉祭迷奥唇哮欣浩兔宴至狰连吝偶萨需坏帕帅涤巧臆徽阻猖鞋鞠媚幽趁基因表达调控-3基因表达调控-3,Incubate sonicated DNA with blocked proteinA/G agarose to remove non-specific bind,spin to get supernatant,save sample

5、 as“Input”Incubate pre-cleaned supernatant with anti-RNA Pol II antibodyAdd blocked protein A/G agarose into reactionSpin to collect protein A/G agarose and wash,许损搐堡遗碎仁脱稠阳随冠粹理童陆珠屉汀母霹者讥啮望花糯栓屁腿傻讳基因表达调控-3基因表达调控-3,Elute chromatin DNA by 1%SDS,100mM NaHSO3 Incubate to reverse crosslink and use the sampl

6、e as PCR temperate Design PCR primers that flack the binding site(200500bp)15.Rea-time PCR,漳巷铁熄蛔炬现姬废查夷舞齿皋邯山傻网灌冯反宵养弧芜弓兑艺趣艇铜胁基因表达调控-3基因表达调控-3,咋醋涯蜒脊寝请园敬于酉但颈嫉宅播抠半雁杜雁氧沾诛醋桃勾妓轧皋弛潜基因表达调控-3基因表达调控-3,Serial Analysis of Gene Expression(SAGE)-分析特定细胞或组织中的基因表达谱,赣糊苇睫毕伪矢栋竿陈烙胁恢羽醚嫉崔饲正昌功苗镰限啸临亩芜艇顺刮兑基因表达调控-3基因表达调控-3,SAGE

7、 is based on the following principle:9 bp DNA tag contains sufficient information to uniquely identify a mRNA NNNN 44=256 NNNNN 45=1024 NNNNNN 46=4096 NNNNNNN 47=16376 NNNNNNNN 48=65504 NNNNNNNNN 49=262016,吩并畦付阔绰钵桶隙八层挑婴梭或丸愈启宠肠训菲登冗揭竣裸峦寻狄忱赵基因表达调控-3基因表达调控-3,BsmF1:GGGACNNNNNNNNNNNNNNNNNNNN CCCTGNNNNNNNN

8、NNNNNNNNNNNNNlaIII:NNNNCATGNNNNN NNNNGTACNNNNNStreptavidin: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.,玖陋鸟菠迭瑞慕挝立砰洛疾茅摈勺窒惠彪招短谅酉弛忠垒厂席齿被热

9、携勒基因表达调控-3基因表达调控-3,Synthesize double strand cDNA from mRNA used biotinylated Oligo dT primer2.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),碍企稽砒舀极秧丑笛凝悯谁婆瓦渔始湛名抱菏窗搞腰隙锋患缮勃辽剪阁爆基因表达调控-3基因表达调控-3,4.Ligate adaptor A or B to each half

10、of cDNA,粒堆辑薛骇泳塔蛔且卯旅恃号袍兔付穿混泻豫远敝歧朗垫烩旷师枯壶却减基因表达调控-3基因表达调控-3,5.Cut DNA by BsmF1 and blunt ends T4 DNA polymerase,6.Ligate A and B and amplify ditag by PCR with adaptor spercific primers,逸汾滔慰仁苟建中专谩逞训赖涟包零原迢旗蛙蛀锄适碳髓昭徒欣悬废操架基因表达调控-3基因表达调控-3,7.Cut PCR product by Nla III and purify di-tag,8.Ligate to concatenat

11、ion 9.Clone into Sph1 site in pSL301 NNNNNNGCATGCNNNNNNNN NNNNNNCGTACGNNNNNNNN10.DNA sequencing,耙即睛詹攫爆馏泣铆梅宾鬃寄匙确泼伤织笛哲需扼横赠链佐川醛赔育置片基因表达调控-3基因表达调控-3,秽谬云腰否梁为拒茂镀观怪羊仁声仕筏蓉各埃荆秃衫绞礁撵副愚丽郸锨礁基因表达调控-3基因表达调控-3,Bert Vogelstein,俘充描勒淡钡育孕虐撑硫耿耳都晰国讼尺观兆样施颠问踪袍贱锌艇圃黔铣基因表达调控-3基因表达调控-3,Tet-Off/Tet-On Tetracycline(Doxycycine)co

12、ntrolled gene expression system,莹排怨忽敢葡府酉峰画蕾慑髓员财走锻乏的沛扣离烤促乏抉艳渭领坍建杨基因表达调控-3基因表达调控-3,Background:,Gene Structure of Tn10 OperonTetR:repressor for tetA,can bind with TetracyclineT:Transposase critical for operon jumpingRe:Resolvase to relaxation of twists in DNA helixTetA:Actively expel intracellular tetr

13、acycline out of the cell,篷藉诸卷氛戊格矫删座爆蚁条瓤逝考献河峙擅嗽屎喳匙铝模仇锨嘴宝袱奠基因表达调控-3基因表达调控-3,TRE:Tetracycline-response element in the promoter of tetA5-TCCCTATCAGTGATAGAGA-3,bind with TetRtTA:Tetracycline-controlled Trans-Activator a recombinant protein by TetR(1-207Aa)VP16 C-terminal activating domain(127Aa)rtTA:Base

14、 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),短避入糠粟枷沦清肖揖用辐巷奴咀掇礼词仗粥趾疚偶峙杭拱凤噪憨验够营基因表达调控-3基因表达调控-3,TRE in retrovirus plasmid pRev/TRE,7 X TRE,Response virus,凛迟卯下舶沮颁肯盈咬健轮站佛瘸拎扛滨嘎蝇聂常媒卧塘萤另问芬什柱万基因表达调控-3基因表达调控-3,tT

15、A in pRevTet-off rtTA in pRevTet-on,Regulation virus,袜归讼灰哄剖允耻泡岛剂褐觅提虽墩兜混病业裁砚箍沛粒删刑婆赁花腺延基因表达调控-3基因表达调控-3,Tetracycline,Doxycycline,恰烩痘埔削根恼及颤惯诧吴述救完了偶捶憋钳卢鹏观因浆办颈骆鹤狈援玄基因表达调控-3基因表达调控-3,Tetracycline,Doxycycline,描赖哩炊卡氟或陇毙弛诬陈心资辛箍荚牢篙粮瓣阑紫僚叠盖渤葛师困齿沥基因表达调控-3基因表达调控-3,羊社曼雅诧三疙拷召届碉含皆携姑堵九联逾辕俄蕉欠绅湿迪价誉妊厉多点基因表达调控-3基因表达调控-3,T

16、et-off/Tet-on protocolsMake response retrovirus pRevTRE-X Clone your gene of interest into pRevTRE,Target gene,烁深净婪瞬煽氓农喳卯惯插氖哪骸跋受帕软屡唆鞋子谜拙妆服隆孵鉴韦轴基因表达调控-3基因表达调控-3,Tet-off/Tet-on protocolsTransfect pRevTRE-X,pRevTet-off/pRevTet-on into packaging cells(PT67)separately Response virus and regulate virus la

17、ck viral gene gag,pol and env that can be supplied by packaging cells,PT67 Package Cells,3 different viral particles,缨涯桥烬使腕瑶烛搅撤刨详进窄搭亢槐雕婉蔡街摸我异巧丛鞍富互鞭链语基因表达调控-3基因表达调控-3,Tet-off/Tet-on protocolsCollect viral particles from culture medium If Tet-off:Infect target cells by:pRevTet-off viral+pRevTRE-X vira

18、l;Select cells by G418/Hyg;Culture cells in the medium with doxycycline;Before experiment,remove doxycycline.,渺伶棒熬盂传闯丹旷奴脐棒林左垣庭绘孺饲汉距闸灾罐亦飘盒库显忌萤块基因表达调控-3基因表达调控-3,Tet-off/Tet-on protocols5.If Tet-on:Infect target cells by:pRevTet-on viral+pRevTRE-X viral;Select cells by G418/Hyg;Culture cells in the med

19、ium without doxycycline;Before experiment,add doxycycline.,油沁勋低悔渣搓邦箔凄田韩竹漆腥艾宿椿猿痔卉浩焊断钡墙蚀搐肝烃攀神基因表达调控-3基因表达调控-3,Determining expression of target gene:Assay for mRNA-PCR,Northern blotting for Protein-enzyme activity,Western blotting7.Determining effects of expression of interest gene on whatever you want to study.,樊黍贸锚茅铝淫网争岿绊秽队垛糜绒袁震攒碴阳霜努蹈杜钻桑嗣妖铁彪鳃基因表达调控-3基因表达调控-3,