掀起Polyplus轉(zhuǎn)染風(fēng)暴——產(chǎn)品專員培訓(xùn)JPolyplus-transfection轉(zhuǎn)染方法介紹：顯微注射法電穿孔法

(Amaxa)Biolistic顆粒傳遞法

（基因槍粒子轟擊法）磷酸鈣法陽(yáng)離子脂質(zhì)體法（Invitrogen，Roche，Qiagen）陽(yáng)離子聚合物

(Sunma梭華，凱基，polyplus)物理方法化學(xué)方法病毒介導(dǎo)法

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Whydoweneedtransfectionreagent?DNA:highlyanionicandhydrophilicmacromolecule

Noionicinteraction++++++++++Cationicmacromolecule++++++CationicReagentMembrane:anionicandhydrophobic----------HeparanSulfateProteoglycanes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PolyPlus-transfection是法國(guó)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(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。第一部分產(chǎn)品介紹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產(chǎn)品系列一Protein&antibody轉(zhuǎn)染:-------PULSin蛋白轉(zhuǎn)染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protein轉(zhuǎn)染難題

MassChargePurityStability3DstructureRelease

ActivityxkDapI=xx%hoursordaysGlobular,random,well-definedstructureFreeproteinincytoplasmDNAandRNA=anionicpolymersProtein=morecomplexmolecules蛋白轉(zhuǎn)染技術(shù)原理PULSin?:

cationicamphiphilic-basedformulation蛋白轉(zhuǎn)染操作步驟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PULSin?轉(zhuǎn)染單抗HeLacellsanti-NPC-(AF?488)1μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyfluorescencemicroscopy8hourspost-delivery.

LocalizedfluorescencearoundnuclearmembraneEfficientdeliveryoffunctionalAb抗體轉(zhuǎn)染PULSin?轉(zhuǎn)染多抗HeLacellsanti-giantin-(AF?488)0.5μlAb4μlPULSin?Conditions:Livingcellswereanalyzedbyconfocalmicroscopy17hourspost-delivery.PlasmamembraneswerestainedwithConcanavalinA-rhodamine.

EfficientdeliveryoffunctionalAb抗體轉(zhuǎn)染

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-rhodaminederivative

EfficientdeliveryofPeptidesPULSin?已轉(zhuǎn)染的不同細(xì)胞類型R-PE:1μg/PULSin?:4μl

Deliverytoawidevarietyofcells蛋白轉(zhuǎn)染PULSin?已轉(zhuǎn)染的細(xì)胞及轉(zhuǎn)染效率蛋白轉(zhuǎn)染R-phycoerythrinR-phycoerythrinR-phycoerythrinPULSin?:蛋白/抗體/多肽列表蛋白轉(zhuǎn)染優(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PULSin?的競(jìng)爭(zhēng)對(duì)手蛋白轉(zhuǎn)染Chariot

(ActiveMotif)BioPorter(GTS)ProteoJuice(Merck)產(chǎn)品系列二體外SiRNA轉(zhuǎn)染:-------INTERFERinsiRNA轉(zhuǎn)染選擇、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)iRNA介導(dǎo)基因沉默機(jī)制

siRNAsiRNAexpressionplasmidCleavagebyDicersiRNAssiRNAassociatewithRISCCleavageofmRNAbyRISCRISCdsRNADicercleavageofdsRNAHairpinsiRNACellmembraneNuclearmembraneProteinXINTERFERin?jetPEI?siRNA轉(zhuǎn)染實(shí)驗(yàn)操作

siRNA(1nM)inserum-freemedium1AddINTERFERin?Vortexandincubate10minatr.t.31234AddtowellcontainingcellsincompletemediumIncubatefor48hoursat37°C56Genesilencingassay6542Protocolforcellsin24-wellplateINTERFERin?siRNA轉(zhuǎn)染反向操作——HTS操作

StandartprotocolReverseprotocolPlatecells24hpriortotransfectionDilutesiRNAsAddINTERFERin?VortexIncubate10minDistributeintowellsDistributesiRNAsoruselibrariesAddINTERFERin?MixIncubate10minAddcells96-wellplateDay1Day2Day1Day3PlateTransfectAssayPlateandtransfectDay4AssaysiRNA轉(zhuǎn)染反向轉(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siRNAdeliverywithINTERFERin?

CaSkicells(24-wellplate)LaminA/CImmunofluorescenceassay1nMsiRNAsiRNA轉(zhuǎn)染基因沉默效率基因沉默效率GreatsilencingdowntopicomolarsiRNAconcentration

A549-GL3Luccells(24-wellplate)Luciferase70%92%siRNA轉(zhuǎn)染細(xì)胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?L1INTERFERin?L2siRNA轉(zhuǎn)染成功轉(zhuǎn)染的細(xì)胞系SilencingefficiencywithINTERFERin?invariouscelllinessiRNA轉(zhuǎn)染原代細(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siRNA轉(zhuǎn)染競(jìng)爭(zhēng)對(duì)手Lipofectamine2000（Invitrogen）Oligofectamine（Invitrogen）HiPerFect（Qiagen）SiLentFect（BioRad）

Invitrogenisthelargest

siRNAtransfectionmarketQiagenisadangerousoutsiderwithHiPerFectsiRNA轉(zhuǎn)染INTERFERinvsLipofectamine2000INTERFERin

vs

OligofectamineINTERFERin

vsHiPerFect/SiLentFectINTERFERin?與HiPerFect/SiLentFect

基因沉默效率

A549-GL3Luc(24-wellplate)LuciferaseINTERFERin?與HiPerFect/SiLentFect

細(xì)胞毒性

A549-GL3Luc(24-wellplate)1nMsiRNANocellulartoxicityobservedwithINTERFERin?HiPerFectINTERFERin?SiLentFect?體內(nèi)DNA&siRNA轉(zhuǎn)染:

------invivojetPEI產(chǎn)品系列三體內(nèi)轉(zhuǎn)染invivojetPEI

vs

VirusesinvivojetPEI:easytobeusednotimmunogenicnottoxiccandeliveredoligonucleotidesuptoverylargeDNA… …butalsosiRNA體內(nèi)轉(zhuǎn)染Efficiency/specificity++++++++++Virusin

vivo-jetPEI?genesize(kbp)3-30>400YesnoneImmuneresponseYes(L2–L3)noneLabrequirement體內(nèi)轉(zhuǎn)染invivojetPEIvsVirusesDifferentroutesVariousanimals

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)染產(chǎn)品系列四DNAinvitro

transfection:

------invitrojetPEI體外DNA轉(zhuǎn)染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：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polyethylenimine(PEI)NH=-CH2-CH2-NH-=43DaammoniumgroupsforDNAcondensationbufferingaminegroupsbranchedPEIlinearPEI1H+/amine3579pH0.5nnjetPEI?體外DNA轉(zhuǎn)染DNAjetPEICationiccomplexesnucleusProtonSpongecytoplasmEndocytosisinvitroDNAdelivery體外DNA轉(zhuǎn)染NHHNOHnmHCl-H3CjetPEI1μl=7.5nmolesofaminefunctionDNA1μg=3nmolesofphosphategroupnumber

of

nitrogen

residues

of

jetPEInumber

of

DNA

phosphates=NPComplexesFormation2μlofjetPEIfor1μgofDNAatN/P=5InvitroDNAdeliveryjetPEI?:實(shí)驗(yàn)操作+serum2μlofjetPEIfor1μgofDNA1mlofjetPEI=500transfectionsin24-wellplates or=2500transfectionsin96-wellplates體外DNA轉(zhuǎn)染HeLacells1μgofpCMVLuc,N/P5ComplexesFormationEffectofincubationtimeSameefficiency體外DNA轉(zhuǎn)染HeLaHEK293NIH3T3?-galactosidaseBHKN/P=3N/P=5controlpCMVLacZReproductibilityCellstransfectedin24-wellplateswithjetPEI(N/P=5)witha?-galactosidase-expressingplasmid(pCMVLacZ).ExpressionwasvisualizedbyX-galstainingafter24h.

60–80%60–80%40–60%jetPEI:轉(zhuǎn)染效率體外DNA轉(zhuǎn)染細(xì)胞特異性jetPEI轉(zhuǎn)