細胞轉(zhuǎn)染試劑產(chǎn)品銷售專員培訓資料詳解演示文稿

細胞轉(zhuǎn)染試劑產(chǎn)品銷售專員培訓資料詳解演示文稿當前第1頁\共有68頁\編于星期四\7點（優(yōu)選）細胞轉(zhuǎn)染試劑產(chǎn)品銷售專員培訓資料當前第2頁\共有68頁\編于星期四\7點

轉(zhuǎn)染的應(yīng)用：基因組功能研究：基因表達調(diào)控，蛋白功能，信號轉(zhuǎn)導和藥物篩選研究建立細胞系：轉(zhuǎn)基因細胞模型、永生性細胞株建立轉(zhuǎn)基因動物模型基因治療研究和應(yīng)用蛋白功能和定位研究重組蛋白生產(chǎn)及蛋白藥物研究當前第3頁\共有68頁\編于星期四\7點Whydoweneedtransfectionreagent?DNA:highlyanionicandhydrophilicmacromoleculeNoionicinteraction++++++++++Cationicmacromolecule++++++CationicReagentMembrane:anionicandhydrophobic----------HeparanSulfateProteoglycanes當前第4頁\共有68頁\編于星期四\7點細胞轉(zhuǎn)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(zhuǎn)染當前第5頁\共有68頁\編于星期四\7點PolyPlus-transfection是法國一家專門致力于研發(fā)和銷售轉(zhuǎn)染試劑的生物技術(shù)公司PolyPlus-transfection公司簡介

PolyPlus-transfection提供體內(nèi)和體外轉(zhuǎn)染產(chǎn)品，包括基因、寡核苷酸、siRNA及蛋白轉(zhuǎn)染產(chǎn)品，可進行瞬時和穩(wěn)定的轉(zhuǎn)染及重組蛋白質(zhì)制備。當前第6頁\共有68頁\編于星期四\7點第一部分產(chǎn)品介紹當前第7頁\共有68頁\編于星期四\7點InVitroInVivosiRNAtransfectionStandardtransfectionINTERFERin?jetSI?-ENDOTraffickingandvisualizationjetSI?-FluojetSI?-ENDO-Fluo產(chǎn)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當前第8頁\共有68頁\編于星期四\7點產(chǎn)品系列

Protein&antibody轉(zhuǎn)染

------PULSinSiRNA體外轉(zhuǎn)染

------INTERFERin

DNA&SiRNA體內(nèi)轉(zhuǎn)染------invivojetPEIDNA體外轉(zhuǎn)染

------invitrojetPEI當前第9頁\共有68頁\編于星期四\7點產(chǎn)品系列一Protein&antibody轉(zhuǎn)染:-------PULSin蛋白轉(zhuǎn)染當前第10頁\共有68頁\編于星期四\7點Protein&antibody轉(zhuǎn)染應(yīng)用：細胞內(nèi)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(nèi)檢測蛋白轉(zhuǎn)染當前第11頁\共有68頁\編于星期四\7點protein轉(zhuǎn)染難題

MassChargePurityStability3DstructureReleaseActivityxkDapI=xx%hoursordaysGlobular,random,well-definedstructureFreeproteinincytoplasmDNAandRNA=anionicpolymersProtein=morecomplexmolecules蛋白轉(zhuǎn)染當前第12頁\共有68頁\編于星期四\7點技術(shù)原理PULSin?:

cationicamphiphilic-basedformulation蛋白轉(zhuǎn)染當前第13頁\共有68頁\編于星期四\7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(zhuǎn)染當前第14頁\共有68頁\編于星期四\7點R-phycoerythrinNIH-3T3cellsConditions:Livingcellswereanalyzedbyfluorescencemicroscopy16hourspost-delivery.pI=5.5MW=240kDaPULSin轉(zhuǎn)染蛋白質(zhì)1μgR-PE + + -

4μlPULSin?

+ - -DiffusefluorescenceEfficientdeliveryofproteinwithPULSin?蛋白轉(zhuǎn)染當前第15頁\共有68頁\編于星期四\7點PULSin?轉(zhuǎn)染單抗HeLacellsanti-NPC-(AF?488)1μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyfluorescencemicroscopy8hourspost-delivery.LocalizedfluorescencearoundnuclearmembraneEfficientdeliveryoffunctionalAb抗體轉(zhuǎn)染當前第16頁\共有68頁\編于星期四\7點PULSin?轉(zhuǎn)染多抗HeLacellsanti-giantin-(AF?488)0.5μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyconfocalmicroscopy17hourspost-delivery.PlasmamembraneswerestainedwithConcanavalinA-rhodamine.EfficientdeliveryoffunctionalAb抗體轉(zhuǎn)染

當前第17頁\共有68頁\編于星期四\7點PULSin?轉(zhuǎn)染多肽多肽轉(zhuǎn)染HeLacellsConditions:DeliveryofPep-A(StreptococcusTPEBepitope,),intoHeLacells.ComplexeswereformedwithPep-A(1μg,lissamine-rhodaminederivative,Sigma)andPULSin?(4μl).Observationwascarriedout16hpost-delivery.Pep-A(StreptococcusTPEBepitope)16aa，lissamine-rhodaminederivativeEfficientdeliveryofPeptides當前第18頁\共有68頁\編于星期四\7點PULSin?已轉(zhuǎn)染的不同細胞類型R-PE:1μg/PULSin?:4μlDeliverytoawidevarietyofcells蛋白轉(zhuǎn)染當前第19頁\共有68頁\編于星期四\7點PULSin?已轉(zhuǎn)染的細胞及轉(zhuǎn)染效率蛋白轉(zhuǎn)染R-phycoerythrinR-phycoerythrinR-phycoerythrin當前第20頁\共有68頁\編于星期四\7點PULSin?:蛋白/抗體/多肽列表蛋白轉(zhuǎn)染當前第21頁\共有68頁\編于星期四\7點優(yōu)勢：

新新技術(shù)——活細胞中進行Protein/Ab轉(zhuǎn)染高轉(zhuǎn)染效率

無細胞毒性

即用型溶液

操作簡單PULSin?優(yōu)勢蛋白轉(zhuǎn)染當前第22頁\共有68頁\編于星期四\7點PULSin?的競爭對手蛋白轉(zhuǎn)染Chariot

(ActiveMotif)BioPorter(GTS)ProteoJuice(Merck)當前第23頁\共有68頁\編于星期四\7點產(chǎn)品系列二體外SiRNA轉(zhuǎn)染:-------INTERFERinsiRNA轉(zhuǎn)染當前第24頁\共有68頁\編于星期四\7點選擇、設(shè)計、合成siRNA雙鏈化學合成siRNA質(zhì)粒表達siRNA轉(zhuǎn)染siRNA到靶細胞瞬時表達——化學合成siRNA，質(zhì)粒表達siRNA穩(wěn)定表達——質(zhì)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(shè)計當前第25頁\共有68頁\編于星期四\7點iRNA介導基因沉默機制

siRNAsiRNAexpressionplasmidCleavagebyDicersiRNAssiRNAassociatewithRISCCleavageofmRNAbyRISCRISCdsRNADicercleavageofdsRNAHairpinsiRNACellmembraneNuclearmembraneProteinXINTERFERin?jetPEI?siRNA轉(zhuǎn)染當前第26頁\共有68頁\編于星期四\7點實驗操作

siRNA(1nM)inserum-freemedium1AddINTERFERin?Vortexandincubate10minatr.t.31234AddtowellcontainingcellsincompletemediumIncubatefor48hoursat37°C56Genesilencingassay6542Protocolforcellsin24-wellplateINTERFERin?siRNA轉(zhuǎn)染當前第27頁\共有68頁\編于星期四\7點反向操作——HTS操作

StandartprotocolReverseprotocolPlatecells24hpriortotransfectionDilutesiRNAsAddINTERFERin?VortexIncubate10minDistributeintowellsDistributesiRNAsoruselibrariesAddINTERFERin?MixIncubate10minAddcells96-wellplateDay1Day2Day1Day3PlateTransfectAssayPlateandtransfectDay4AssaysiRNA轉(zhuǎn)染當前第28頁\共有68頁\編于星期四\7點反向轉(zhuǎn)染基因沉默效率

A549-GL3LucLuciferase96-wellplate反向轉(zhuǎn)染仍能得到極高的基因沉默效率siRNA轉(zhuǎn)染當前第29頁\共有68頁\編于星期四\7點siRNAdeliverywithINTERFERin?

CaSkicells(24-wellplate)LaminA/CImmunofluorescenceassay1nMsiRNAsiRNA轉(zhuǎn)染基因沉默效率當前第30頁\共有68頁\編于星期四\7點基因沉默效率GreatsilencingdowntopicomolarsiRNAconcentration

A549-GL3Luccells(24-wellplate)Luciferase70%92%siRNA轉(zhuǎn)染當前第31頁\共有68頁\編于星期四\7點細胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?L1INTERFERin?L2siRNA轉(zhuǎn)染當前第32頁\共有68頁\編于星期四\7點成功轉(zhuǎn)染的細胞系SilencingefficiencywithINTERFERin?invariouscelllinessiRNA轉(zhuǎn)染當前第33頁\共有68頁\編于星期四\7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(zhì)細胞人原代纖維原細胞82%siRNA轉(zhuǎn)染當前第34頁\共有68頁\編于星期四\7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(zhuǎn)染中的效率高siRNA轉(zhuǎn)染當前第35頁\共有68頁\編于星期四\7點siRNA轉(zhuǎn)染競爭對手Lipofectamine2000（Invitrogen）Oligofectamine（Invitrogen）HiPerFect（Qiagen）SiLentFect（BioRad）

Invitrogenisthelargest

siRNAtransfectionmarketQiagenisadangerousoutsiderwithHiPerFectsiRNA轉(zhuǎn)染當前第36頁\共有68頁\編于星期四\7點INTERFERinvsLipofectamine2000當前第37頁\共有68頁\編于星期四\7點INTERFERin

vs

Oligofectamine當前第38頁\共有68頁\編于星期四\7點INTERFERin

vsHiPerFect/SiLentFect當前第39頁\共有68頁\編于星期四\7點INTERFERin?與HiPerFect/SiLentFect

基因沉默效率

A549-GL3Luc(24-wellplate)Luciferase當前第40頁\共有68頁\編于星期四\7點INTERFERin?與HiPerFect/SiLentFect

細胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?HiPerFectINTERFERin?SiLentFect?當前第41頁\共有68頁\編于星期四\7點體內(nèi)DNA&siRNA轉(zhuǎn)染:

------invivojetPEI產(chǎn)品系列三體內(nèi)轉(zhuǎn)染當前第42頁\共有68頁\編于星期四\7點invivojetPEI

vs

VirusesinvivojetPEI:easytobeusednotimmunogenicnottoxiccandeliveredoligonucleotidesuptoverylargeDNA… …butalsosiRNA體內(nèi)轉(zhuǎn)染當前第43頁\共有68頁\編于星期四\7點Efficiency/specificity++++++++++Virusin

vivo-jetPEI?genesize(kbp)3-30>400YesnoneImmuneresponseYes(L2–L3)noneLabrequirement體內(nèi)轉(zhuǎn)染invivojetPEIvsViruses當前第44頁\共有68頁\編于星期四\7點DifferentroutesVariousanimals

Intravenous靜脈Intratumoral瘤內(nèi)Intracerebral腦室Intraperitonal腹膜Intratracheal氣管灌注Instillation…吸入MouseRatDuckGuineapigMonkey…體內(nèi)轉(zhuǎn)染的路徑和動物…towardsHumanapplication體內(nèi)轉(zhuǎn)染當前第45頁\共有68頁\編于星期四\7點體內(nèi)轉(zhuǎn)染的基因表達分布情況O.Freund活小鼠體內(nèi)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(nèi)轉(zhuǎn)染體內(nèi)轉(zhuǎn)染當前第46頁\共有68頁\編于星期四\7點InRatsH19-DTA（H19-白喉毒素A基因）質(zhì)粒與invivo-jetPEI復合物轉(zhuǎn)入大鼠膀胱。

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

Ohana,P.,etal.GeneTherMolBiol(2004)8,181-192DecreaseinbladderweightInhibitionoftumorgrowthinvivo-jetPEI轉(zhuǎn)染對膀胱癌的治療體內(nèi)轉(zhuǎn)染當前第47頁\共有68頁\編于星期四\7點InHumanH19-DTAplasmid/invivo-jetPEI?復合物轉(zhuǎn)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(zhuǎn)染對膀胱癌的治療體內(nèi)轉(zhuǎn)染當前第48頁\共有68頁\編于星期四\7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(zhuǎn)染siRNAandpCMVLuc與invivo-jetPEI復合物轉(zhuǎn)染對熒光素酶肺部表達起明顯的抑制作用。體內(nèi)轉(zhuǎn)染當前第49頁\共有68頁\編于星期四\7點Urban-kleinetal.,GeneTherapy,1-6,2004C-erb2/neu(HER-2)receptorinvivo-jetPEI：--體內(nèi)基因沉默HER-2downregulationTumorgrowthinhibitionduring18daysi.p.inathymicnudemiceSubcutaneoustumorxenograftsSKOV-3(ovariancarcinomacell)A)NakedHER-2specificsiRNA(0.6nmole)B)HER-2siRNA/PEIpolyplexes體內(nèi)轉(zhuǎn)染當前第50頁\共有68頁\編于星期四\7點產(chǎn)品系列四DNAinvitro

transfection:

------invitrojetPEI體外DNA轉(zhuǎn)染當前第51頁\共有68頁\編于星期四\7點DNA轉(zhuǎn)染過程體外DNA轉(zhuǎn)染提取質(zhì)粒DNA或寡核苷酸選擇和培養(yǎng)細胞系轉(zhuǎn)染瞬時轉(zhuǎn)染穩(wěn)定轉(zhuǎn)染：抗性篩選檢測外源基因細胞內(nèi)表達的實驗流程：當前第52頁\共有68頁\編于星期四\7點DNA轉(zhuǎn)染試劑+H3NO+ONH3CCH3H3CDOTMA(Lipofectin?)MonocationiclipidsPolycationiclipids++NONOHNHNH3H2NTransfectam?CationicpolymersPAMAMDendrimerOHONHONHOH2NNHRNHRNHRnPolylysineandderivativesNNNNNNNNNNH2NNH2NH2NH2NH2NH2NH2H2NH2NH2NH2NNH2012GenerationsNHNNHNHNNHNNHNHNHNNH3+NHNHNNHNHNNNHNH2NH+3HN+3HNNH3+NH3+NH3+Polyethylenimine(PEI)體外DNA轉(zhuǎn)染當前第53頁\共有68頁\編于星期四\7點polyethylenimine(PEI)NH=-CH2-CH2-NH-=43DaammoniumgroupsforDNAcondensationbufferingaminegroupsbranchedPEIlinearPEI1H+/amine3579pH0.5nnjetPEI?體外DNA轉(zhuǎn)染當前第54頁\共有68頁\編于星期四\7點DNAjetPEICationiccomplexesnucleusProtonSpongecytoplasmEndocytosisinvitroDNAdelivery體外DNA轉(zhuǎn)染NHHNOHnmHCl-H3C當前第55頁\共有68頁\編于星期四\7點jetPEI1μl=7.5nmolesofaminefunctionDNA1μg=3nmolesofphosphategroupnumber

of

nitrogen

residues

of

jetPEInumber

of

DNA

phosphates=NPComplexesFormation2μlofjetPEIfor1μgofDNAatN/P=5InvitroDNAdelivery當前第56頁\共有68頁\編于星期四\7點jetPEI?:實驗操作+serum2μlofjetPEIfor1μgofDNA1mlofjetPEI=500transfectionsin24-wellplates or=2500transfectionsin96-wellplates體外DNA轉(zhuǎn)染當前第57頁\共有68頁\編于星期四\7點HeLacells1μgofpCMVLuc,N/P5ComplexesFormationEffectofincubationtimeSameefficiency體外DNA轉(zhuǎn)染當前第58頁\共有68頁\編于星期四\7點HeLaHEK293NIH3T3?-galactosidaseBHKN/P=3N/P=5controlpCMVLacZReproductibilityCellstransfectedin24-wellplateswithjetPEI