研究生分子生物学工作基础新基因研究路线.pptVIP

PhenotypicalReadoutsgrowthanddevelopmentAnimalgrowthanddevelopmentSystem/organ/tissuegrowthandproliferationmorphologyandotherbehaviorallychangesmetabolicchangesCellularlevelchangesintargetgeneexpressionchangesinsignaltransductionpathwayseffectsondownstreamgenesMolecularLevelStealth?RNAi

—最有效进行基因沉默的RNAi双链体StealthRNAi是经化学修饰合成的RNAi双链体，可产生高特异性的有效的基因表达抑制。它克服了非修饰

siRNA分子的限制，因此可以产生更清晰的结果。。避免脱靶效应—StealthRNAi的正义链是经过化学修饰的，仅反义链能够可进入RNAi通路，解决了敏感

的脱靶问题。

。血清和细胞培养中的高稳定性—在进行动物研究时无需验证新的试剂，节约您的时间。

。消除非特异性效应—避免了细胞应激反应通路的激活，避免产生非特异结果。*Manyproteinsfunctionwithpartnersorascomponentsoflargemulti-proteincomplexes.Understandingtheseinteractionsiscriticaltounderstandingbiologicalpathwaysandcellularfunction.Glutathione-STransferase(GST)pull-down(1)isanimportanttoolforvalidationofsuspectedprotein:proteininteractionsandforidentificationofinteractingpartners(2–5).InGSTpull-downassaysaGST-fusionbaitproteinisexpressedinE.coli,thenboundtoglutathione(GSH)-coupledparticlesandusedtoaffinitypurifyanyproteins(prey)thatinteractwiththebaitfromapoolofproteinsinsolution(Figure1).Preyproteinscomefrommultiplesourcesincludingcelllysates,purifiedproteinsandcell-freeexpressionsystems.Cell-freesystemseasilyexpressavarietyofpreyproteinsandmapthedomainnecessaryforasuccessfulinteraction.*

LossofFunction

InhibitionofactivityInhibitionofexpressionInhibitionofActivitySpecificinhibitorsDominantnegativesSpecificantibodiesHistoneDeacetylase(HDAC)inhibitorsDominantNegativeAmutationwhosegeneproductadverselyaffectsthenormal,wild-typegeneproductwithinthesamecell.Thisusuallyoccursiftheproductcanstillinteractwiththesameelementsasthewild-typeproduct,butblocksomeaspectofitsfunction.Examples:

1.Amutationinatranscriptionfactorthatremovestheactivationdomain,butsti