2013前沿技术讲座幻灯rna干扰文库及基因组

siRNAvs (mRNAvssiRNAvs humanbrachyuryoranyothergene

Brachyuryknockdowninα-

BySuleiPax6knockdowninpax6pax61.5relativerelativemRNA0.5

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•7•8•7•7•750%40%30%20%10%0%pSicoR-mTOR-50%40%30%20%10%0%pSicoR-mTOR-shRNA-pSicoR-mTOR-shRNA-pSicoR-mTOR-shRNA-pSicoR-mTOR-shRNA-pSicoR-2-shRNApSicoRintactclone Abortedclone100%100%90%percentagepercentageof70%60%mTOR- (Zhouetal,PNAS,(Zhouetal,PNAS, 激 HumanKinomeshRNA(>700humankinase,3,700shRNAconstructs)

Intracellular

Sox-

Oct-KinomeprovidestheentrypointforhESCchemical

humankinomeshRNA smids

2293T

Colonyspread(16kinasewasidentified,unpublishedsiRNA：Dharmacon公司SmartpoolshRNA：SigmapLKOAmbionsiRNAtargetfinderOligoMaker建议：尽量选用已 shRNA48hsiRNAshRNA48h mRNA水平：最好选用定量蛋白水平：多个时间点的Westernblotting ’OE“挽救”干扰表型实验’55CodingCoding 列withwith

PickupSingle

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Second

Selectedknock-downstablecelllines(MILLERJC,Naturebiotechnology,2011;MOSCOUMJ,Science,BOCHJ,Science,2009；LIT,Nucleicacidsresearch,

名名

SUNN,MolecularbioSystems,CRISPRsclusteredregularlyinterspacedshortpalindromicrepeats WeiC,etal.JGenetGenomics.CRISPRgRNA Guidesequence的接头序列与载体BbsI ZhangLab,NatProtoc.2