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This,,,observation,,,was,,,extended,,,to,,,mammalian,,,somatic,,,cells,,,by,,,Tuschl,,,and,,,coworkers,,,(Elbashir,,,et,,,al.,,,Monahan,,PE,,,Jooss,,K,,,Sands,,MS.,,Through,,,the,,,application,,,of,,,combinatorial,,,RNAi,,,(co-RNAi),,,,multiple,,,shRNA,,,delivery,,,strategies,,,can,,,improve,,,gene,,,knockdown,,,,permit,,,multiple,,,transcripts,,,to,,,be,,,targeted,,,simultaneously,,,,and,,,curtail,,,the,,,emergence,,,of,,,viral,,,escape,,,mutants.,,,A,,,,subset,,,,of,,,,these,,,,shRNAs,,,,is,,,,depicted,,,,in,,,,Figure,,,,1A.,,,,Genes,,,&,,,Dev.,,,If,,the,,genes,,are,,provided,,in,,two,,separate,,vectors,,,use,,5,,μg,,of,,each,,vector.10Viral,,supernatant,,is,,infectious,,and,,should,,be,,treated,,with,,appropriate,,precautions.11For,,viruses,,encoding,,a,,fluorescent,,marker,,gene,,,such,,as,,GFP,,,293T,,cells,,can,,be,,checked,,microscopically,,at,,this,,point,,to,,ensure,,transfection,,efficiency.,,We,,,find,,,that,,,roughly,,,50%,,,of,,,both,,,siRNAs,,,and,,,shRNAs,,,are,,,competent,,,for,,,suppressing,,,gene,,,expression.,,,We,,,,therefore,,,,explored,,,,the,,,,use,,,,of,,,,alternative,,,,promoter,,,,strategies,,,,in,,,,an,,,,effort,,,,to,,,,produce,,,,more,,,,defined,,,,hairpin,,,,RNAs.,,,,

6.,,,2001;,,Ketting,,et,,al.,,2001a).,,The,,,,average,,,,of,,,,three,,,,independent,,,,experiments,,,,is,,,,shown;,,,,error,,,,bars,,,,indicate,,,,standard,,,,deviation.,,,,HEK,,,,293T,,,,cells,,,,were,,,,transfected,,,,with,,,,plasmids,,,,as,,,,in,,,,Figure,,,,2.,,,,The,,293T,,plates,,can,,now,,be,,bleached,,and,,discarded,,according,,to,,approved,,biosafety,,procedures.Use,,viral,,supernatant,,fresh,,or,,aliquot,,and,,freeze,,at,,−80C,,for,,at,,least,,6,,months,,to,,1,,year.3.4.,,Updated,,,,plasmid,,,,information,,,,can,,,,be,,,,obtained,,,,at,,,,Plasmids,,,,expressing,,,,hairpinRNAs,,,,The,,,,U6,,,,promoter,,,,region,,,,from,,,,265,,,,to,,,,+1,,,,was,,,,amplified,,,,by,,,,PCR,,,,,adding,,,,5,,,,KpnI,,,,and,,,,3,,,,EcoRV,,,,sites,,,,for,,,,cloning,,,,into,,,,pBSSK+.,,,,The,,,,presence,,,,of,,,,non-base-paired,,,,guanosine,,,,residues,,,,at,,,,the,,,,5,,,,end,,,,of,,,,siRNAs,,,,significantly,,,,alters,,,,the,,,,predicted,,,,end,,,,structure,,,,and,,,,abolishes,,,,siRNA,,,,activity.,,,,

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