細(xì)胞轉(zhuǎn)染試劑產(chǎn)品銷(xiāo)售專(zhuān)員培訓(xùn)資料詳解演示文稿

細(xì)胞轉(zhuǎn)染試劑產(chǎn)品銷(xiāo)售專(zhuān)員培訓(xùn)資料詳解演示文稿目前一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)（優(yōu)選）細(xì)胞轉(zhuǎn)染試劑產(chǎn)品銷(xiāo)售專(zhuān)員培訓(xùn)資料目前二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)

轉(zhuǎn)染的應(yīng)用：基因組功能研究：基因表達(dá)調(diào)控，蛋白功能，信號(hào)轉(zhuǎn)導(dǎo)和藥物篩選研究建立細(xì)胞系：轉(zhuǎn)基因細(xì)胞模型、永生性細(xì)胞株建立轉(zhuǎn)基因動(dòng)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)及蛋白藥物研究目前三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)Whydoweneedtransfectionreagent?DNA:highlyanionicandhydrophilicmacromoleculeNoionicinteraction++++++++++Cationicmacromolecule++++++CationicReagentMembrane:anionicandhydrophobic----------HeparanSulfateProteoglycanes目前四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)細(xì)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)染目前五頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)PolyPlus-transfection是法國(guó)一家專(zhuān)門(mén)致力于研發(fā)和銷(xiāo)售轉(zhuǎn)染試劑的生物技術(shù)公司PolyPlus-transfection公司簡(jiǎn)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(jìn)行瞬時(shí)和穩(wěn)定的轉(zhuǎn)染及重組蛋白質(zhì)制備。目前六頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)第一部分產(chǎn)品介紹目前七頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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目前八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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目前九頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)產(chǎn)品系列一Protein&antibody轉(zhuǎn)染:-------PULSin蛋白轉(zhuǎn)染目前十頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)Protein&antibody轉(zhuǎn)染應(yīng)用：細(xì)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(xì)胞化學(xué)在活細(xì)胞內(nèi)檢測(cè)蛋白轉(zhuǎn)染目前十一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)protein轉(zhuǎn)染難題

MassChargePurityStability3DstructureReleaseActivityxkDapI=xx%hoursordaysGlobular,random,well-definedstructureFreeproteinincytoplasmDNAandRNA=anionicpolymersProtein=morecomplexmolecules蛋白轉(zhuǎn)染目前十二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)技術(shù)原理PULSin?:

cationicamphiphilic-basedformulation蛋白轉(zhuǎn)染目前十三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)操作步驟21For24-wellplate:細(xì)胞融合度：70-80%稀釋蛋白：1μgofprotein/100μlHepes1加4μl

PULSin?2孵育15min3洗細(xì)胞，加900μl無(wú)血清培養(yǎng)基4加protein/PULSin復(fù)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(cè)6蛋白轉(zhuǎn)染目前十四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)R-phycoerythrinNIH-3T3cellsConditions:Livingcellswereanalyzedbyfluorescencemicroscopy16hourspost-delivery.pI=5.5MW=240kDaPULSin轉(zhuǎn)染蛋白質(zhì)1μgR-PE + + -

4μlPULSin?

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

目前十七頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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目前十八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)PULSin?已轉(zhuǎn)染的不同細(xì)胞類(lèi)型R-PE:1μg/PULSin?:4μlDeliverytoawidevarietyofcells蛋白轉(zhuǎn)染目前十九頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)PULSin?已轉(zhuǎn)染的細(xì)胞及轉(zhuǎn)染效率蛋白轉(zhuǎn)染R-phycoerythrinR-phycoerythrinR-phycoerythrin目前二十頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)PULSin?:蛋白/抗體/多肽列表蛋白轉(zhuǎn)染目前二十一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)優(yōu)勢(shì)：

新新技術(shù)——活細(xì)胞中進(jìn)行Protein/Ab轉(zhuǎn)染高轉(zhuǎn)染效率

無(wú)細(xì)胞毒性

即用型溶液

操作簡(jiǎn)單PULSin?優(yōu)勢(shì)蛋白轉(zhuǎn)染目前二十二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)PULSin?的競(jìng)爭(zhēng)對(duì)手蛋白轉(zhuǎn)染Chariot

(ActiveMotif)BioPorter(GTS)ProteoJuice(Merck)目前二十三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)產(chǎn)品系列二體外SiRNA轉(zhuǎn)染:-------INTERFERinsiRNA轉(zhuǎn)染目前二十四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)選擇、設(shè)計(jì)、合成siRNA雙鏈化學(xué)合成siRNA質(zhì)粒表達(dá)siRNA轉(zhuǎn)染siRNA到靶細(xì)胞瞬時(shí)表達(dá)——化學(xué)合成siRNA，質(zhì)粒表達(dá)siRNA穩(wěn)定表達(dá)——質(zhì)粒表達(dá)siRNA分析檢測(cè)RNA干擾作用蛋白水平:WB/IF/FCmRNA水平:實(shí)時(shí)定量RT-PCR，northernblottingsiRNA基因沉默實(shí)驗(yàn)設(shè)計(jì)目前二十五頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)iRNA介導(dǎo)基因沉默機(jī)制

siRNAsiRNAexpressionplasmidCleavagebyDicersiRNAssiRNAassociatewithRISCCleavageofmRNAbyRISCRISCdsRNADicercleavageofdsRNAHairpinsiRNACellmembraneNuclearmembraneProteinXINTERFERin?jetPEI?siRNA轉(zhuǎn)染目前二十六頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)實(shí)驗(yàn)操作

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

StandartprotocolReverseprotocolPlatecells24hpriortotransfectionDilutesiRNAsAddINTERFERin?VortexIncubate10minDistributeintowellsDistributesiRNAsoruselibrariesAddINTERFERin?MixIncubate10minAddcells96-wellplateDay1Day2Day1Day3PlateTransfectAssayPlateandtransfectDay4AssaysiRNA轉(zhuǎn)染目前二十八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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)染目前二十九頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)siRNAdeliverywithINTERFERin?

CaSkicells(24-wellplate)LaminA/CImmunofluorescenceassay1nMsiRNAsiRNA轉(zhuǎn)染基因沉默效率目前三十頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)基因沉默效率GreatsilencingdowntopicomolarsiRNAconcentration

A549-GL3Luccells(24-wellplate)Luciferase70%92%siRNA轉(zhuǎn)染目前三十一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)細(xì)胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?L1INTERFERin?L2siRNA轉(zhuǎn)染目前三十二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)成功轉(zhuǎn)染的細(xì)胞系SilencingefficiencywithINTERFERin?invariouscelllinessiRNA轉(zhuǎn)染目前三十三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)原代細(xì)胞的高效沉默

24-wellplateGAPDH(甘油醛-3-磷酸脫氫酶)BranchedDNAassay96%人原代肝實(shí)質(zhì)細(xì)胞人原代纖維原細(xì)胞82%siRNA轉(zhuǎn)染目前三十四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)INTERFERin優(yōu)勢(shì)低濃度siRNA（甚至pmol級(jí)別）就能達(dá)到高效的基因沉默在很多細(xì)胞系中，基因沉默效率可達(dá)到90％以上在原代細(xì)胞中也能達(dá)到有效的基因沉默效率無(wú)細(xì)胞毒性不受血清和抗生素的影響操作簡(jiǎn)單價(jià)格便宜在反向操作轉(zhuǎn)染中的效率高siRNA轉(zhuǎn)染目前三十五頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)siRNA轉(zhuǎn)染競(jìng)爭(zhēng)對(duì)手Lipofectamine2000（Invitrogen）Oligofectamine（Invitrogen）HiPerFect（Qiagen）SiLentFect（BioRad）

Invitrogenisthelargest

siRNAtransfectionmarketQiagenisadangerousoutsiderwithHiPerFectsiRNA轉(zhuǎn)染目前三十六頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)INTERFERinvsLipofectamine2000目前三十七頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)INTERFERin

vs

Oligofectamine目前三十八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)INTERFERin

vsHiPerFect/SiLentFect目前三十九頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)INTERFERin?與HiPerFect/SiLentFect

基因沉默效率

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

細(xì)胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?HiPerFectINTERFERin?SiLentFect?目前四十一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)體內(nèi)DNA&siRNA轉(zhuǎn)染:

------invivojetPEI產(chǎn)品系列三體內(nèi)轉(zhuǎn)染目前四十二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)invivojetPEI

vs

VirusesinvivojetPEI:easytobeusednotimmunogenicnottoxiccandeliveredoligonucleotidesuptoverylargeDNA… …butalsosiRNA體內(nèi)轉(zhuǎn)染目前四十三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)Efficiency/specificity++++++++++Virusin

vivo-jetPEI?genesize(kbp)3-30>400YesnoneImmuneresponseYes(L2–L3)noneLabrequirement體內(nèi)轉(zhuǎn)染invivojetPEIvsViruses目前四十四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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(dòng)物…towardsHumanapplication體內(nèi)轉(zhuǎn)染目前四十五頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)體內(nèi)轉(zhuǎn)染的基因表達(dá)分布情況O.Freund活小鼠體內(nèi)熒光素酶表達(dá)的熒光檢測(cè)圖象（用含50μgpCMVluc和invivo-jetPEI復(fù)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)染目前四十六頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)InRatsH19-DTA（H19-白喉毒素A基因）質(zhì)粒與invivo-jetPEI復(fù)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(duì)膀胱癌的治療體內(nèi)轉(zhuǎn)染目前四十七頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)InHumanH19-DTAplasmid/invivo-jetPEI?復(fù)合物轉(zhuǎn)染對(duì)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(duì)膀胱癌的治療體內(nèi)轉(zhuǎn)染目前四十八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)活體小鼠尾靜脈注射（24小時(shí)后的熒光檢測(cè)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(fù)合物轉(zhuǎn)染對(duì)熒光素酶肺部表達(dá)起明顯的抑制作用。體內(nèi)轉(zhuǎn)染目前四十九頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)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)染目前五十頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)產(chǎn)品系列四DNAinvitro

transfection:

------invitrojetPEI體外DNA轉(zhuǎn)染目前五十一頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)DNA轉(zhuǎn)染過(guò)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(xì)胞系轉(zhuǎn)染瞬時(shí)轉(zhuǎn)染穩(wěn)定轉(zhuǎn)染：抗性篩選檢測(cè)外源基因細(xì)胞內(nèi)表達(dá)的實(shí)驗(yàn)流程：目前五十二頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)DNA轉(zhuǎn)染試劑+H3NO+ONH3CCH3H3CDOTMA(Lipofectin?)MonocationiclipidsPolycationiclipids++NONOHNHNH3H2NTransfectam?CationicpolymersPAMAMDendrimerOHONHONHOH2NNHRNHRNHRnPolylysineandderivativesNNNNNNNNNNH2NNH2NH2NH2NH2NH2NH2H2NH2NH2NH2NNH2012GenerationsNHNNHNHNNHNNHNHNHNNH3+NHNHNNHNHNNNHNH2NH+3HN+3HNNH3+NH3+NH3+Polyethylenimine(PEI)體外DNA轉(zhuǎn)染目前五十三頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)polyethylenimine(PEI)NH=-CH2-CH2-NH-=43DaammoniumgroupsforDNAcondensationbufferingaminegroupsbranchedPEIlinearPEI1H+/amine3579pH0.5nnjetPEI?體外DNA轉(zhuǎn)染目前五十四頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)DNAjetPEICationiccomplexesnucleusProtonSpongecytoplasmEndocytosisinvitroDNAdelivery體外DNA轉(zhuǎn)染NHHNOHnmHCl-H3C目前五十五頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)jetPEI1μl=7.5nmolesofaminefunctionDNA1μg=3nmolesofphosphategroupnumber

of

nitrogen

residues

of

jetPEInumber

of

DNA

phosphates=NPComplexesFormation2μlofjetPEIfor1μgofDNAatN/P=5InvitroDNAdelivery目前五十六頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)jetPEI?:實(shí)驗(yàn)操作+serum2μlofjetPEIfor1μgofDNA1mlofjetPEI=500transfectionsin24-wellplates or=2500transfectionsin96-wellplates體外DNA轉(zhuǎn)染目前五十七頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)HeLacells1μgofpCMVLuc,N/P5ComplexesFormationEffectofincubationtimeSameefficiency體外DNA轉(zhuǎn)染目前五十八頁(yè)\總數(shù)六十八頁(yè)\編于十九點(diǎn)HeLaHEK293NIH3T3?-galactosidaseBHKN/P=3N/P=5controlpCMVLacZReproductibilityCellstransfectedin24-wellplateswithje