細胞轉染試劑產品銷售專員培訓資料詳解演示文稿

細胞轉染試劑產品銷售專員培訓資料詳解演示文稿目前一頁\總數六十八頁\編于十九點（優(yōu)選）細胞轉染試劑產品銷售專員培訓資料目前二頁\總數六十八頁\編于十九點

轉染的應用：基因組功能研究：基因表達調控，蛋白功能，信號轉導和藥物篩選研究建立細胞系：轉基因細胞模型、永生性細胞株建立轉基因動物模型基因治療研究和應用蛋白功能和定位研究重組蛋白生產及蛋白藥物研究目前三頁\總數六十八頁\編于十九點Whydoweneedtransfectionreagent?DNA:highlyanionicandhydrophilicmacromoleculeNoionicinteraction++++++++++Cationicmacromolecule++++++CationicReagentMembrane:anionicandhydrophobic----------HeparanSulfateProteoglycanes目前四頁\總數六十八頁\編于十九點細胞轉染難易比較Adherent>Suspension Problem: E.g.:MembraneTHP1,Jurkat…Fastdividingcell>nondividingcell Problem: E.g.:AccesstothenucleusNeurons,…Cellline>Primarycell Problem: E.g.:MoresensitiveEpithelialprimarycells…體外轉染目前五頁\總數六十八頁\編于十九點PolyPlus-transfection是法國一家專門致力于研發(fā)和銷售轉染試劑的生物技術公司PolyPlus-transfection公司簡介

PolyPlus-transfection提供體內和體外轉染產品，包括基因、寡核苷酸、siRNA及蛋白轉染產品，可進行瞬時和穩(wěn)定的轉染及重組蛋白質制備。目前六頁\總數六十八頁\編于十九點第一部分產品介紹目前七頁\總數六十八頁\編于十九點InVitroInVivosiRNAtransfectionStandardtransfectionINTERFERin?jetSI?-ENDOTraffickingandvisualizationjetSI?-FluojetSI?-ENDO-Fluo產品介紹DNAtransfectionStandardtransfection

invivo-jetPEI?Ligand-conjugatedreagents

invivo-jetPEI?-Galinvivo-jetPEI?-ManTraffickingandvisualization

invivo-jetPEI?-Fluoinvivo-jetPEI?-biotinDNAtransfectionInvitroInvivoStandardtransfection

jetPEI?Cell-specifictransfectionjetPEI?-GaljetPEI?-MacrophagejetPEI?-HUVECjetPEI?-RGDTraffickingandvisualization

jetPEI?-FluojetPEI?-biotinSyntheticmediumtransfectionFecturin?ProteindeliveryProteinandantibody

PULSin?invivo-jetPEI?MousebraininjectionjetSI?10mMsiRNAtransfection目前八頁\總數六十八頁\編于十九點產品系列

Protein&antibody轉染

------PULSinSiRNA體外轉染

------INTERFERin

DNA&SiRNA體內轉染------invivojetPEIDNA體外轉染

------invitrojetPEI目前九頁\總數六十八頁\編于十九點產品系列一Protein&antibody轉染:-------PULSin蛋白轉染目前十頁\總數六十八頁\編于十九點Protein&antibody轉染應用：細胞內定位研究蛋白水平的干擾研究抗體功能封閉的研究疾病治療的研究替代免疫細胞化學在活細胞內檢測蛋白轉染目前十一頁\總數六十八頁\編于十九點protein轉染難題

MassChargePurityStability3DstructureReleaseActivityxkDapI=xx%hoursordaysGlobular,random,well-definedstructureFreeproteinincytoplasmDNAandRNA=anionicpolymersProtein=morecomplexmolecules蛋白轉染目前十二頁\總數六十八頁\編于十九點技術原理PULSin?:

cationicamphiphilic-basedformulation蛋白轉染目前十三頁\總數六十八頁\編于十九點操作步驟21For24-wellplate:細胞融合度：70-80%稀釋蛋白：1μgofprotein/100μlHepes1加4μl

PULSin?2孵育15min3洗細胞，加900μl無血清培養(yǎng)基4加protein/PULSin復合物345孵育4h5更換新鮮完全培養(yǎng)基6分析蛋白活性或檢測6蛋白轉染目前十四頁\總數六十八頁\編于十九點R-phycoerythrinNIH-3T3cellsConditions:Livingcellswereanalyzedbyfluorescencemicroscopy16hourspost-delivery.pI=5.5MW=240kDaPULSin轉染蛋白質1μgR-PE + + -

4μlPULSin?

+ - -DiffusefluorescenceEfficientdeliveryofproteinwithPULSin?蛋白轉染目前十五頁\總數六十八頁\編于十九點PULSin?轉染單抗HeLacellsanti-NPC-(AF?488)1μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyfluorescencemicroscopy8hourspost-delivery.LocalizedfluorescencearoundnuclearmembraneEfficientdeliveryoffunctionalAb抗體轉染目前十六頁\總數六十八頁\編于十九點PULSin?轉染多抗HeLacellsanti-giantin-(AF?488)0.5μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyconfocalmicroscopy17hourspost-delivery.PlasmamembraneswerestainedwithConcanavalinA-rhodamine.EfficientdeliveryoffunctionalAb抗體轉染

目前十七頁\總數六十八頁\編于十九點PULSin?轉染多肽多肽轉染HeLacellsConditions:DeliveryofPep-A(StreptococcusTPEBepitope,),intoHeLacells.ComplexeswereformedwithPep-A(1μg,lissamine-rhodaminederivative,Sigma)andPULSin?(4μl).Observationwascarriedout16hpost-delivery.Pep-A(StreptococcusTPEBepitope)16aa，lissamine-rhodaminederivativeEfficientdeliveryofPeptides目前十八頁\總數六十八頁\編于十九點PULSin?已轉染的不同細胞類型R-PE:1μg/PULSin?:4μlDeliverytoawidevarietyofcells蛋白轉染目前十九頁\總數六十八頁\編于十九點PULSin?已轉染的細胞及轉染效率蛋白轉染R-phycoerythrinR-phycoerythrinR-phycoerythrin目前二十頁\總數六十八頁\編于十九點PULSin?:蛋白/抗體/多肽列表蛋白轉染目前二十一頁\總數六十八頁\編于十九點優(yōu)勢：

新新技術——活細胞中進行Protein/Ab轉染高轉染效率

無細胞毒性

即用型溶液

操作簡單PULSin?優(yōu)勢蛋白轉染目前二十二頁\總數六十八頁\編于十九點PULSin?的競爭對手蛋白轉染Chariot

(ActiveMotif)BioPorter(GTS)ProteoJuice(Merck)目前二十三頁\總數六十八頁\編于十九點產品系列二體外SiRNA轉染:-------INTERFERinsiRNA轉染目前二十四頁\總數六十八頁\編于十九點選擇、設計、合成siRNA雙鏈化學合成siRNA質粒表達siRNA轉染siRNA到靶細胞瞬時表達——化學合成siRNA，質粒表達siRNA穩(wěn)定表達——質粒表達siRNA分析檢測RNA干擾作用蛋白水平:WB/IF/FCmRNA水平:實時定量RT-PCR，northernblottingsiRNA基因沉默實驗設計目前二十五頁\總數六十八頁\編于十九點iRNA介導基因沉默機制

siRNAsiRNAexpressionplasmidCleavagebyDicersiRNAssiRNAassociatewithRISCCleavageofmRNAbyRISCRISCdsRNADicercleavageofdsRNAHairpinsiRNACellmembraneNuclearmembraneProteinXINTERFERin?jetPEI?siRNA轉染目前二十六頁\總數六十八頁\編于十九點實驗操作

siRNA(1nM)inserum-freemedium1AddINTERFERin?Vortexandincubate10minatr.t.31234AddtowellcontainingcellsincompletemediumIncubatefor48hoursat37°C56Genesilencingassay6542Protocolforcellsin24-wellplateINTERFERin?siRNA轉染目前二十七頁\總數六十八頁\編于十九點反向操作——HTS操作

StandartprotocolReverseprotocolPlatecells24hpriortotransfectionDilutesiRNAsAddINTERFERin?VortexIncubate10minDistributeintowellsDistributesiRNAsoruselibrariesAddINTERFERin?MixIncubate10minAddcells96-wellplateDay1Day2Day1Day3PlateTransfectAssayPlateandtransfectDay4AssaysiRNA轉染目前二十八頁\總數六十八頁\編于十九點反向轉染基因沉默效率

A549-GL3LucLuciferase96-wellplate反向轉染仍能得到極高的基因沉默效率siRNA轉染目前二十九頁\總數六十八頁\編于十九點siRNAdeliverywithINTERFERin?

CaSkicells(24-wellplate)LaminA/CImmunofluorescenceassay1nMsiRNAsiRNA轉染基因沉默效率目前三十頁\總數六十八頁\編于十九點基因沉默效率GreatsilencingdowntopicomolarsiRNAconcentration

A549-GL3Luccells(24-wellplate)Luciferase70%92%siRNA轉染目前三十一頁\總數六十八頁\編于十九點細胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?L1INTERFERin?L2siRNA轉染目前三十二頁\總數六十八頁\編于十九點成功轉染的細胞系SilencingefficiencywithINTERFERin?invariouscelllinessiRNA轉染目前三十三頁\總數六十八頁\編于十九點原代細胞的高效沉默

24-wellplateGAPDH(甘油醛-3-磷酸脫氫酶)BranchedDNAassay96%人原代肝實質細胞人原代纖維原細胞82%siRNA轉染目前三十四頁\總數六十八頁\編于十九點INTERFERin優(yōu)勢低濃度siRNA（甚至pmol級別）就能達到高效的基因沉默在很多細胞系中，基因沉默效率可達到90％以上在原代細胞中也能達到有效的基因沉默效率無細胞毒性不受血清和抗生素的影響操作簡單價格便宜在反向操作轉染中的效率高siRNA轉染目前三十五頁\總數六十八頁\編于十九點siRNA轉染競爭對手Lipofectamine2000（Invitrogen）Oligofectamine（Invitrogen）HiPerFect（Qiagen）SiLentFect（BioRad）

Invitrogenisthelargest

siRNAtransfectionmarketQiagenisadangerousoutsiderwithHiPerFectsiRNA轉染目前三十六頁\總數六十八頁\編于十九點INTERFERinvsLipofectamine2000目前三十七頁\總數六十八頁\編于十九點INTERFERin

vs

Oligofectamine目前三十八頁\總數六十八頁\編于十九點INTERFERin

vsHiPerFect/SiLentFect目前三十九頁\總數六十八頁\編于十九點INTERFERin?與HiPerFect/SiLentFect

基因沉默效率

A549-GL3Luc(24-wellplate)Luciferase目前四十頁\總數六十八頁\編于十九點INTERFERin?與HiPerFect/SiLentFect

細胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?HiPerFectINTERFERin?SiLentFect?目前四十一頁\總數六十八頁\編于十九點體內DNA&siRNA轉染:

------invivojetPEI產品系列三體內轉染目前四十二頁\總數六十八頁\編于十九點invivojetPEI

vs

VirusesinvivojetPEI:easytobeusednotimmunogenicnottoxiccandeliveredoligonucleotidesuptoverylargeDNA… …butalsosiRNA體內轉染目前四十三頁\總數六十八頁\編于十九點Efficiency/specificity++++++++++Virusin

vivo-jetPEI?genesize(kbp)3-30>400YesnoneImmuneresponseYes(L2–L3)noneLabrequirement體內轉染invivojetPEIvsViruses目前四十四頁\總數六十八頁\編于十九點DifferentroutesVariousanimals

Intravenous靜脈Intratumoral瘤內Intracerebral腦室Intraperitonal腹膜Intratracheal氣管灌注Instillation…吸入MouseRatDuckGuineapigMonkey…體內轉染的路徑和動物…towardsHumanapplication體內轉染目前四十五頁\總數六十八頁\編于十九點體內轉染的基因表達分布情況O.Freund活小鼠體內熒光素酶表達的熒光檢測圖象（用含50μgpCMVluc和invivo-jetPEI復合物的400μl5%葡萄糖溶液尾部注射Balb/Cmice）

Liver30000Tumor2000KidneyL300000LungR20000000KidneyR115000OvaryL210000LungL16000000OvaryR150000CourtesyJL.CollandO.Freundinvivo-jetPEI體內轉染體內轉染目前四十六頁\總數六十八頁\編于十九點InRatsH19-DTA（H19-白喉毒素A基因）質粒與invivo-jetPEI復合物轉入大鼠膀胱。

NBT-IIcells 50μgDNAatD+4&D+8 D+11ratsweresacrified

Ohana,P.,etal.GeneTherMolBiol(2004)8,181-192DecreaseinbladderweightInhibitionoftumorgrowthinvivo-jetPEI轉染對膀胱癌的治療體內轉染目前四十七頁\總數六十八頁\編于十九點InHumanH19-DTAplasmid/invivo-jetPEI?復合物轉染對人膀胱癌的治療Ohana,P.,etal.GeneTherMolBiol(2004)8,181-192(D)Video-cystoscopyperformedbeforethetreatmentandthreeweeksafterthecompletionofthe6thtreatment,showingalargenecroticareareplacingthetumorregion(E).Treatmentshows>75%tumorregressioninvivo-jetPEI轉染對膀胱癌的治療體內轉染目前四十八頁\總數六十八頁\編于十九點活體小鼠尾靜脈注射（24小時后的熒光檢測圖）24hourslater,theluciferasegeneexpressionwasmonitoredbybioluminescentimagingoflivemiceusingacooledCCDcamera.DNAalone(GL2-Luc)DNA(GL2-Luc)+GL2-Luc-siRNA,10μgDNA(GL2-Luc)+GL3-Luc-siRNA,10μgColl,Freund,Erbacherinvivo-jetPEI:DNA-siRNA共轉染siRNAandpCMVLuc與invivo-jetPEI復合物轉染對熒光素酶肺部表達起明顯的抑制作用。體內轉染目前四十九頁\總數六十八頁\編于十九點Urban-kleinetal.,GeneTherapy,1-6,2004C-erb2/neu(HER-2)receptorinvivo-jetPEI：--體內基因沉默HER-2downregulationTumorgrowthinhibitionduring18daysi.p.inathymicnudemiceSubcutaneoustumorxenograftsSKOV-3(ovariancarcinomacell)A)NakedHER-2specificsiRNA(0.6nmole)B)HER-2siRNA/PEIpolyplexes體內轉染目前五十頁\總數六十八頁\編于十九點產品系列四DNAinvitro

transfection:

------invitrojetPEI體外DNA轉染目前五十一頁\總數六十八頁\編于十九點DNA轉染過程體外DNA轉染提取質粒DNA或寡核苷酸選擇和培養(yǎng)細胞系轉染瞬時轉染穩(wěn)定轉染：抗性篩選檢測外源基因細胞內表達的實驗流程：目前五十二頁\總數六十八頁\編于十九點DNA轉染試劑+H3NO+ONH3CCH3H3CDOTMA(Lipofectin?)MonocationiclipidsPolycationiclipids++NONOHNHNH3H2NTransfectam?CationicpolymersPAMAMDendrimerOHONHONHOH2NNHRNHRNHRnPolylysineandderivativesNNNNNNNNNNH2NNH2NH2NH2NH2NH2NH2H2NH2NH2NH2NNH2012GenerationsNHNNHNHNNHNNHNHNHNNH3+NHNHNNHNHNNNHNH2NH+3HN+3HNNH3+NH3+NH3+Polyethylenimine(PEI)體外DNA轉染目前五十三頁\總數六十八頁\編于十九點polyethylenimine(PEI)NH=-CH2-CH2-NH-=43DaammoniumgroupsforDNAcondensationbufferingaminegroupsbranchedPEIlinearPEI1H+/amine3579pH0.5nnjetPEI?體外DNA轉染目前五十四頁\總數六十八頁\編于十九點DNAjetPEICationiccomplexesnucleusProtonSpongecytoplasmEndocytosisinvitroDNAdelivery體外DNA轉染NHHNOHnmHCl-H3C目前五十五頁\總數六十八頁\編于十九點jetPEI1μl=7.5nmolesofaminefunctionDNA1μg=3nmolesofphosphategroupnumber

of

nitrogen

residues

of

jetPEInumber

of

DNA

phosphates=NPComplexesFormation2μlofjetPEIfor1μgofDNAatN/P=5InvitroDNAdelivery目前五十六頁\總數六十八頁\編于十九點jetPEI?:實驗操作+serum2μlofjetPEIfor1μgofDNA1mlofjetPEI=500transfectionsin24-wellplates or=2500transfectionsin96-wellplates體外DNA轉染目前五十七頁\總數六十八頁\編于十九點HeLacells1μgofpCMVLuc,N/P5ComplexesFormationEffectofincubationtimeSameefficiency體外DNA轉染目前五十八頁\總數六十八頁\編于十九點HeLaHEK293NIH3T3?-galactosidaseBHKN/P=3N/P=5controlpCMVLacZReproductibilityCellstransfectedin24-wellplateswithje