遺伝子導入技術 - Bio-Rad

1 386 387 390 390 391 398 386 DNA RNA 1986 Transfection TechnologiesPage PlantsBacteriaFungiAnimalsLipid TransfectionsiLentFect lipid reagent for RNAi388 TransFectin lipid reagent389 ElectroporationGene Pulser MXcell 392 Gene Pulser Xcell 394 * * Gene Pulser Xcell CE 394 * * Gene Pulser Xcell PC 394 MicroPulser 396 Biolistic Particle DeliveryHelios gene gun 399 PDS-1000/He 401 PDS-1000/He Hepta 401 * Mammalian Cells Gene KnockdownsAdherent Cells, TrypsinizedAdherent Cells, AttachedOrganelles (chloroplasts, mitochondria)Plant Cell Suspension CulturesPlant Cell Adherent CulturesPlant Cell Callus or ExplantsPollenLeaves in SituDetached LeavesMeristems in SituDetached MeristemsGram-Positive BacteriaGram-Negative BacteriaYeastOther FungiSkin or Organs in VivoOrgan CulturesEx Vivo Cells or Tissues ( , blood)

2 Suspension Cell Cultures IonsGeneCell MembraneABCGasSampleHelios Gene Gun System Particle Delivery MethodDNA coated microcarrier dioleoylphosphatidylethanolamine DOPE DNA DNA DNA DNA siLentFect Lipid ReagentTransFectin Lipid Reagent RNAi siRNA siRNA(shRNA) A549 BHK HeLa K562 Jurkat Vero Primary Fibroblasts A549 MCF-7 HeLa HUVEC Jurkat Primary Fibroblasts 388 siLentFect Lipid Reagent for RNAi small interfering RNA siRNA siLentFect Lipid Reagent siRNA siRNA siRNA siLentFect Caco-2 siLentFect siRNA MLCK1 Bulletin 5370 siRNA siRNA siLentFect siRNA dsDNA vectors Suggested Reagent Quantities for Different Sizes of Plates/WellsCultureVessel SizeVolume ofPlating of

3 SerumFree *5-20nM50 *5-20nM100 l6 *5-20nM250 *5-20nM500 * l* Carefully aspirate medium 15-60 minutes prior to transfection and add one-half volume of of Cell Lines Successfully Transfected With siLentFect Lipid Reagent*Cell LineCell TypeCell LineCell Type184htrtHuman breast epithelial LNCapHuman prostate adenocarcinoma4T1 Mouse mammary tumor MCF-7 Human breast adenocarcinomaA549 Human lung carcinoma Murine ECMouse endothelial Caco-2 Human colon adenocarcinomaNIH 3T3 Mouse embryo fibroblastCHO-K1 Chinese hamster ovary epithelial PC-3 Human prostate cancerCOS-7SV40-transformed African green monkey kidney Primary fibroblastHuman foreskin fibroblastHEK 293 Human embryonic kidney Primary keratinocyteHuman keratinocyteHeLaHuman cervical adenocarcinomaPrimary ovarianEpithelialHepG2 Human liver carcinomaSVEC4 Vascular endothelialHUVECH uman umbilical vein endothelialVSMCV ascular smooth muscle* Bulletin 5343 Ordering Information 170-3360siLentFect Lipid Reagent, 34,0004 C170-3361siLentFect Lipid Reagent, 53,0004 C170-3362siLentFect Lipid Reagent, 5 168,0004 CMCF-7 siRNA Hoechst siRNAsiLentFect Lipid Reagent siRNA lacZ CHO lacZ siRNA siLentFect Lipid Reagent 24well CHO siRNA siLentFect Lipid Reagent 96well 1well l , %020406080100siLentFect volume, l/well volume, l/well 0 2 10 20 503,0002,5002,0001,5001,0005000 Luciferase activity, RLUsiRNA, nM020406080100 Downregulation, %siRNA.

4 NM 0 2 10 20 50 See Also PCR PCR TransFectin Lipid ReagentTransFectin Lipid Reagent 3 40 90% confluence Suggested Reagent Quantities for Different Sizes of Plates/WellsCultureVessel SizeSurfaceArea (cm2)Volume ofPlating MediaPlasmidDNAV olume of SerumFree g50 g100 l6 g250 g500 l15-20 lExamples of Cell Lines Successfully Transfected With TransFectin Lipid Reagent*Cell LineCell TypeCell LineCell TypeA375 Human malignant melanomaK562 Human bone marrow, chronic myelogenous leukemiaA549 Human lung carcinomaLLC-MK2 Monkey epithelial kidneyACHNH uman renal adenocarcinomaLNCaPHuman prostate adenocarcinomaBHKN ewborn Syrian golden hamster kidney MEFM ouse embryo fibroblastCHOC hinese hamster ovaryNBT-IIRat bladder carcinomaCOS-7SV40-transformed African green monkey kidneyNeuro-2aMouse neuroblastomaCV-1 African green monkey fibroblast kidneyNIH 3T3 Mouse embryo fibroblastHEK 293 Human embryonic kidneySH-SY5 YHuman neuroblastomaHeLaHuman cervical adenocarcinomaSW480 Human colorectal adenocarcinomaHep G2 Human epithelial liver carcinomaVeroAfrican green monkey kidney* Bulletin 5343 Ordering Information 170-3350 TransFectin Lipid Reagent, 29,0004 C170-3351 TransFectin Lipid Reagent, 45,0004 C170-3352 TransFectin Lipid Reagent, 5 147.

5 0004 CTransFectin LipidReagent K562 DNA TransFectin LipidReagent 1well 4 105 cell 24well 24 DNA l l 1 2 3 405 Volume of TransFectin, l 50 l 4 l TransFectin 50 l 1 g DNA TransFectin DNA 20 DNA- DNA- 100 l/24 TransFectin 3 See Also PCR DNA DNA RNA Gene Pulser MXcell Gene Pulser Xcell MicroPulser Gene Pulser Gene Pulser Gene Pulser siRNA DNA Gene Pulser siRNA DNA PBS 1/2 1/3 F Ordering Information

6 165-2676 Gene Pulser 10 29,0004 C165-2677 Gene Pulser 30ml 42,0004 CIonsGeneCell membraneABC A B C DNA See Also Gene Pulser Gene Pulser Xcell Gene Pulser MXcell PulseTrac PulseTrac V0 Voltage, VTime, ms V0eTime, msV0 Voltage, VVttA.

7 2 ( ) V0 V0 37% (R) (C) ( R C) (E kV/cm) V0 B. (t) pulse droop(Vt) 5% BA392 Gene Pulser MXcell Gene Pulser Mxcell 96well 24well 12well 3 1 24 1 1 24 1 2 96well/Exp, Vgrad/Cgrad ABCD1 12 123456789101112 V 350 F 1000 A V(V)

8 100100100200200200300300300400400400BC EFGH1 12 D C 250V 1000 E C( F)20020020035035035050050050010001000100 0 FGH 96well/Sqr, Vgrad, Dgrad ABCD1 12 123456789101112 V 2000 F 1000 20msA V(V)100100100200200200300300300400400400 BC EFGH1 12 D D 250V 2000 F 1000 E D(ms)101010151515202020303030 FGH Opt 96well/Exp, Sqr ABCD1 12 123456789101112 EXPA150V200V250V300V350V450V250V250V250V 250V250V250V350 F350 F350 F350 F350 F350 F200 F250 F350 F500 F750 F1000 FBC EFGH1 12 D SQRE150V200V250V300V350V450V250V250V250V 250V250V250V20ms20ms20ms20ms20ms20ms5ms1 0ms15ms20ms25ms30msFGH 96 Well See Also Gene Pulser MXcell 100-120 VAC 220-240 VAC 50/60Hz 240W EN61010 EMC EN61326 Class A 31 30 14cm V 10 500 2V F 25 2,475 25 F 50 1,500 50 10 10 500V msec 9.

9 10 500V 1 3 10 500V sec 10 10 500V Gene Pulser MXcell Single ShockPod Gene Pulser MXcell Gene Pulser MXcell 96well 24well 12well 3 Gene Pulser MXcell Sample Volume Selection GuideFormatWell VolumeCell ConcentrationNumber ofWell Sets12-well 106 to 1071224-well plate500 800 l5 105 to 8 1062496-well plate100 200 l1 105 to 2 cuvette400 800 l4 105 to 8 106N/AOrdering Information 165-2670 JAGene Pulser MXcell 1,320,000165-2670 JBGene Pulser MXcell Single Shock Pod 1,440,000165-2673 Gene Pulser MXcell Single Shock Pod 250,000165-2681 Gene Pulser MXcell 96well 38,000165-2681B05 Gene Pulser MXcell 96well (5 ) 180,000165-2682 Gene Pulser MXcell 24well 38,000165-2682B05 Gene Pulser MXcell 24well (5 ) 180,000165-2683 Gene Pulser MXcell 12well 38,000165-2683B05 Gene Pulser MXcell 12well (5 ) 180,000 Gene Pulser MXcell Single ShockPod394 Mammalian CellsBacterial CellsFungal CellsCHO E.

10 Coli S. cerevisiaeCOS7 A. tumefaciensP. pastoris3T3 P. aeruginosa C. albicans293 S. aureusS. pombeHeLaB. cereus D. discoideumBHK21 S. pyrogenesA549 L. plantarumCV1K562HL60 JurkatHuT78 Gene Pulser Xcell Gene Pulser Xcell Gene Pulser Gene PulserII Gene Pulser Gene Pulser Xcell PulseTrac 144 100 PC module 3000V ShockPod Cuvette ChamberPC CE See Also Gene Pulser Xcell 100-120 VAC 220-240 VAC 50/60Hz