細胞轉(zhuǎn)染試劑產(chǎn)品銷售專員培訓資料PPT課件

掀起Polyplus轉(zhuǎn)染風暴 產(chǎn)品專員培訓 J Polyplus 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 磷酸鈣法陽離子脂質(zhì)體法 Invitrogen Roche Qiagen 陽離子聚合物 Sunma梭華 凱基 polyplus 物理方法 化學方法 病毒介導法 轉(zhuǎn)染的應用 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 細胞轉(zhuǎn)染難易比較 Adherent SuspensionProblem E g MembraneTHP1 Jurkat Fastdividingcell nondividingcellProblem E g AccesstothenucleusNeurons Cellline PrimarycellProblem E g MoresensitiveEpithelialprimarycells 體外轉(zhuǎn)染 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 InVitro InVivo siRNAtransfection StandardtransfectionINTERFERin jetSI ENDOTraffickingandvisualizationjetSI FluojetSI ENDO Fluo 產(chǎn)品介紹 DNAtransfection Standardtransfectioninvivo jetPEI Ligand conjugatedreagentsinvivo jetPEI Galinvivo jetPEI ManTraffickingandvisualizationinvivo jetPEI Fluoinvivo jetPEI biotin DNAtransfection Invitro Invivo StandardtransfectionjetPEI Cell specifictransfectionjetPEI GaljetPEI MacrophagejetPEI HUVECjetPEI RGDTraffickingandvisualizationjetPEI FluojetPEI biotin SyntheticmediumtransfectionFecturin Proteindelivery ProteinandantibodyPULSin invivo jetPEI MousebraininjectionjetSI 10mM siRNAtransfection 產(chǎn)品系列 Protein antibody轉(zhuǎn)染 PULSinSiRNA體外轉(zhuǎn)染 INTERFERinDNA 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 protein轉(zhuǎn)染難題 MassChargePurityStability3DstructureRelease Activity xkDapI xx hoursordaysGlobular random well definedstructureFreeproteinincytoplasm DNAandRNA anionicpolymers Protein morecomplexmolecules 蛋白轉(zhuǎn)染 技術(shù)原理 PULSin cationicamphiphilic basedformulation 蛋白轉(zhuǎn)染 操作步驟 蛋白轉(zhuǎn)染 R phycoerythrin NIH 3T3cells Conditions Livingcellswereanalyzedbyfluorescencemicroscopy16hourspost delivery pI 5 5MW 240kDa PULSin轉(zhuǎn)染蛋白質(zhì) 1 gR PE 4 lPULSin Diffusefluorescence EfficientdeliveryofproteinwithPULSin 蛋白轉(zhuǎn)染 PULSin 轉(zhuǎn)染單抗 HeLacellsanti NPC AF 488 1 lAb4 lPULSin Conditions Livingcellswereanalyzedbyfluorescencemicroscopy8hourspost delivery Localizedfluorescencearoundnuclearmembrane EfficientdeliveryoffunctionalAb 抗體轉(zhuǎn)染 PULSin 轉(zhuǎn)染多抗 HeLacellsanti giantin AF 488 0 5 lAb4 lPULSin Conditions Livingcellswereanalyzedbyconfocalmicroscopy17hourspost delivery PlasmamembraneswerestainedwithConcanavalinA rhodamine EfficientdeliveryoffunctionalAb 抗體轉(zhuǎn)染 PULSin 轉(zhuǎn)染多肽 多肽轉(zhuǎn)染 HeLacells Pep A StreptococcusTPEBepitope 16aa lissamine rhodaminederivative EfficientdeliveryofPeptides PULSin 已轉(zhuǎn)染的不同細胞類型 R PE 1 g PULSin 4 l Deliverytoawidevarietyofcells 蛋白轉(zhuǎn)染 PULSin 已轉(zhuǎn)染的細胞及轉(zhuǎn)染效率 蛋白轉(zhuǎn)染 R phycoerythrin R phycoerythrin R phycoerythrin PULSin 蛋白 抗體 多肽列表 蛋白轉(zhuǎn)染 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 PULSin 的競爭對手 蛋白轉(zhuǎn)染 Chariot ActiveMotif BioPorter GTS ProteoJuice Merck 產(chǎn)品系列二 體外SiRNA轉(zhuǎn)染 INTERFERin siRNA轉(zhuǎn)染 選擇 設計 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 northernblotting siRNA基因沉默實驗設計 iRNA介導基因沉默機制 siRNA siRNAexpressionplasmid CleavagebyDicer siRNAs siRNAassociatewithRISC CleavageofmRNAbyRISC dsRNA DicercleavageofdsRNA HairpinsiRNA Cellmembrane Nuclearmembrane Protein X INTERFERin jetPEI siRNA轉(zhuǎn)染 實驗操作 siRNA 1nM inserum freemedium 1 AddINTERFERin Vortexandincubate10minatr t 3 1 2 3 4 Addtowellcontainingcellsincompletemedium Incubatefor48hoursat37 C 5 6 Genesilencingassay 6 5 4 2 Protocolforcellsin24 wellplate INTERFERin siRNA轉(zhuǎn)染 反向操作 HTS操作 Standartprotocol Reverseprotocol Platecells24hpriortotransfection DilutesiRNAs AddINTERFERin VortexIncubate10min Distributeintowells DistributesiRNAsoruselibraries AddINTERFERin MixIncubate10min Addcells 96 wellplate Day1 Day2 Day1 Day3 Plate Transfect Assay Plateandtransfect Day4 Assay siRNA轉(zhuǎn)染 反向轉(zhuǎn)染基因沉默效率 A549 GL3LucLuciferase 96 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 CImmunofluorescenceassay 1nMsiRNA siRNA轉(zhuǎn)染 基因沉默效率 基因沉默效率 GreatsilencingdowntopicomolarsiRNAconcentration A549 GL3Luccells 24 wellplate Luciferase 70 92 siRNA轉(zhuǎn)染 細胞毒性 A549 GL3Luc 24 wellplate 1nMsiRNA NocellulartoxicityobservedwithINTERFERin L1 INTERFERin L2 siRNA轉(zhuǎn)染 成功轉(zhuǎn)染的細胞系 SilencingefficiencywithINTERFERin invariouscelllines siRNA轉(zhuǎn)染 原代細胞的高效沉默 24 wellplateGAPDH 甘油醛 3 磷酸脫氫酶 BranchedDNAassay 96 人原代肝實質(zhì)細胞 人原代纖維原細胞 82 siRNA轉(zhuǎn)染 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 siRNA轉(zhuǎn)染競爭對手 Lipofectamine2000 Invitrogen Oligofectamine Invitrogen HiPerFect Qiagen SiLentFect BioRad InvitrogenisthelargestsiRNAtransfectionmarketQiagenisadangerousoutsiderwithHiPerFect siRNA轉(zhuǎn)染 INTERFERinvsLipofectamine2000 INTERFERinvsOligofectamine INTERFERinvsHiPerFect SiLentFect INTERFERin 與HiPerFect SiLentFect基因沉默效率 A549 GL3Luc 24 wellplate Luciferase INTERFERin 與HiPerFect SiLentFect細胞毒性 A549 GL3Luc 24 wellplate 1nMsiRNA NocellulartoxicityobservedwithINTERFERin HiPerFect INTERFERin SiLentFect 體內(nèi)DNA siRNA轉(zhuǎn)染 invivojetPEI 產(chǎn)品系列三 體內(nèi)轉(zhuǎn)染 invivojetPEIvsViruses invivojetPEI easytobeusednotimmunogenicnottoxiccandeliveredoligonucleotidesuptoverylargeDNA butalsosiRNA 體內(nèi)轉(zhuǎn)染 體內(nèi)轉(zhuǎn)染 invivojetPEIvsViruses Differentroutes Variousanimals 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 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50 gpCMVluc和invivo jetPEI復合物的400 l5 葡萄糖溶液尾部注射Balb Cmice Liver30000Tumor2000 KidneyL300000 LungR20000000 KidneyR115000 OvaryL210000 LungL16000000 OvaryR150000 CourtesyJL CollandO Freund invivo jetPEI體內(nèi)轉(zhuǎn)染 體內(nèi)轉(zhuǎn)染 InRats H19 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 IIcells50 gDNAatD 4 D 8D 11ratsweresacrified Ohana P etal GeneTherMolBiol 2004 8 181 192 DecreaseinbladderweightInhibitionoftumorgrowth invivo jetPEI轉(zhuǎn)染對膀胱癌的治療 體內(nèi)轉(zhuǎn)染 InHuman H19 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 tumorregression invivo jetPEI轉(zhuǎn)染對膀胱癌的治療 體內(nèi)轉(zhuǎn)染 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 10 g DNA GL2 Luc GL3 Luc siRNA 10 g Coll Freund Erbacher invivo 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 Urban kleinetal GeneTherapy 1 6 2004 C erb2 neu HER 2 receptor invivo jetPEI 體內(nèi)基因沉默 HER 2downregulationTumorgrowthinhibitionduring18days i 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 DNAinvitrotransfection invitrojetPEI 體外DNA轉(zhuǎn)染 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 DNA轉(zhuǎn)染試劑 體外DNA轉(zhuǎn)染 體外DNA轉(zhuǎn)染 DNA jetPEI Cationiccomplexes nucleus ProtonSponge cytoplasm Endocytosis invitroDNAdelivery 體外DNA轉(zhuǎn)染 numberofnitrogenresiduesofjetPEI numberofDNAphosphates ComplexesFormation 2 lofjetPEIfor1 gofDNAatN P 5 InvitroDNAdelivery jetPEI 實驗操作 serum 2 lofjetPEIfor1 gofDNA 1mlofjetPEI 500transfectionsin24 wellplatesor 2500transfectionsin96 wellplates 體外DNA轉(zhuǎn)染 HeLacells 1 gofpCMVLuc N P5 ComplexesFormation Effectofincubationtime 體外DNA轉(zhuǎn)染 HeLa HEK293 NIH3T3 galactosidase BHK N P 3 N P 5 control pCMVLacZ Reproductibility Cellstransfectedin24 wellplateswithjetPEI N P 5 witha galactosidase expressingplasmid pCMVLacZ ExpressionwasvisualizedbyX galstainingafter24h jetPEI 轉(zhuǎn)染效率 體外DNA轉(zhuǎn)染 細胞特異性jetPEI轉(zhuǎn)染產(chǎn)品 jetPEI Macrophage 巨噬細胞 jetPEI Mannose Mannosereceptor MacrophagesjetPEI Hepatocyte 肝實質(zhì)細胞 jetPEI G