EditPro™ Stem Transfection Reagent/代理/现货/报价

　　EditPro™StemTransfectionReagentisoptimizedforgenomeeditinginhumanpluripotentstemcellsandneuralstemcells.ItispossIBLetoco-transfectdifferentsubstratesprovidingtheflexibilityneededforgenomeeditingexperiments.ForexampleEditPro™StemprovidesefficienttransfectionoflargeDNAplasmidssuchasCRISPR/Cas9vectors,RNAsuchasCas9mRNAco-transfectedwithtracr/guideRNA,orproteinandRNAsuchasCas9proteinwithtracr/guideRNA.ItalsoresultsinhighefficiencytransfectionwithTALENsandZFNgeneeditingtools.

　　Viewourpresentation:OptimizingDeliveryofGene-EditingToolsinPluripotentStemCellsandIsolationandExpansionofClonalTargetedLines

　　EditPro™StemTransfectionReagentiseasytouseandcanworkwithvarioushumanpluripotentstemcellcultureconditions(mediaandmatrices).Theefficiencyismaximizedwhentransfectingwiththecellsinadherence,however,whenusedinconjunctionwiththePluriQ™G9™MaintenanceMediumKitwiththecellsplatedonG9™VTNHumanRecombinantasmatrixasprovidedinthePluriQ™G9™GeneEditingSystem.Thereasonfortheboostusingthisculturingformatisthatthecellsgrowmoreasamonolayerandthereforearemoreaccessibletotransfectionresultinginevenandhighefficiencytransfectionwithlowtoxicity.

 

　　Fig.1iPSCellsgrowninPluriQ™G9™MaintenanceMediumonG9™VTNHumanRecombinantasmatrix.

　　EditPro™StemwhenusedtotransfectiPSorhEScellsaspartofthePluriQ™G9™GeneEditingSystemresultsinthehighestefficiencyoftransfectionregardlessofwhattypeofsubstrateisbeingtransfected.Asanexample,weshowtransfectionandexpressionefficiencywithaCas9DNAplasmid,andINDELformationwithtransfectionofmRNAwithtracr/guideRNAorCas9proteinwithtracr/guideRNA.Allmethodsgiveexcellenteditingresults.

　　

 

　　Fig.2HighEfficiencyTransfectionwithastandardCRISPR/Cas9EF1a-GFPDNAconstruct.NCRM-1iPScellstransfectedusingEditProStemwitha~10.5kbplasmidexpressingCas9andGFP.

　　

 

　　Fig.3TransfectionwithEditPro™StemTransfectionReagentResultsinEffectiveEditing.CellsweretransfectedwithCas9mRNA(modified)/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified)orCas9protein/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified).Genome-modificationwasanalyzedusingtheT7Endo1assay.

　　Toallowflexibilityinchoiceofpluripotentstemcellculturesystem,suchasplatingonothermatricesorinothercommercialmedia,weoptimizedtransfectioninsUSPensionusinga"reversetransfection"technique,formaximumtransfectionofallthecellsinculture.Adherentcellsgrownincoloniesonthesematricesdonotevenlytransfectduetolimitedaccessofthereagenttotheinnercellsofthecolony.Figure3illustrateshowco-transfectionofmodifiedGFPmRNAandCas9RNPusingtheEditProStemreagentconsistentlyproduces>90%transfectionefficiencyasmonitoredbyGFPinstemcellsplatedonvitronectininPluriQ™G9™MaintenanceMediumandonGeltrexinmTeSR™1.

　　

 

　　Fig.4TransfectioninSuspension:ExpressionofeGFPmRNAco-deliveredwithCas9Protein,tracrRNAandcrRNAsEmx1inhumanESCsplatedonA.vitronectininPluriQG9MaintenanceMediumorB.GeltrexinmTeSR

　　Usingthetransfectioninsuspensionprotocol,themodifiedCas9mRNA,tracrRNAandamodifiedGFPmRNAwereco-transfected.ThecellswerethenanalyzedbyT7Endo1assaytolookattheeffectivenessofediting.

　　

 

　　Fig.5Cas9mRNATransfectionintoiPSCCellswithEditPro™Stem:200,000cellsweretransfectedinsuspensionusingEditPro™Stemwith250ngCas9mRNA(modified)/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified).Genome-modificationwasanalyzedusingtheT7Endo1assay.

　　

 

　　Fig.6Cas9Protein(RNP)TransfectionofhumaniPSandEScellswithEditPro™Stem:200,000cellsweretransfectedinsuspensionusingEditPro™StemwithCas9protein/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified).Genome-modificationwasanalyzedusingtheT7Endo1assay

　　EditPro™Stemprovidessuperiordeliveryintoneuralstemcells.UsingiPS-derivedhumanneuralstemcellsandtheprotocolfortransfectioninsuspensionwithsubsequentplatingonGeltrexinNeuralX™NSCMediumSupplementedwithGS22™wesee>90%efficiencybyGFPreporterandexcellenteditingusingeitherCas9mRNAorCas9Protein(RNP)asevaluatedbyT7Endo1assay.

　　

 

　　

 

　　Fig.7GeneEditingbyco-TransfectionusingEditPro™StemTransfectionReagentinNeuralStemCells:NeuralStemcellsweretransfectedinsuspensionusingEditPro™Stemwith250ngCas9mRNA(modified)/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified)orCas9protein/gRNA:Emx-1crRNA(Exon2)-tracrRNAoligo/GFPmRNA(modified).GFPwasobservedafter24hours.Genome-modificationwasanalyzedusingtheT7Endo1assay.

　　Features:

　　HighEfficiency–deliveringDNA,RNAand/orprotein

　　Lowtoxicityanddoesnotaffectpluripotency

　　Easytouse–nospecialequipmentrequired

　　CostEffective–effectiveatlowamountsofreagentresultinginlowcost/well

　　TechnicalData

　　EditPro™StemProtocolforPluripotentStemCells

　　EditPro™StemProtocolforNeuralStemCells

　　Publications

　　Kehler,J.,Greco,M.,Martino,V.,Pachiappan,M.,Yokoe,H.,Chen,A.,Yang,M.,Jessee,J.,Gotte,M.,Milanesi,L.,Albertini,A.,Bellipanni,G.,Zucchi,I.,Reinbold,R.A.andGiordano,A.(2016),RNA-GeneratedandGene-EditedInducedPluripotentStemCellsforDiseaseModelingandTherapy.J.Cell.Physiol.2016Sep15.doi:10.1002/jcp.25597.[Epubaheadofprint]