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)

2 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)Suspension Cell Cultures IonsGeneCell MembraneABCGasSampleHelios Gene Gun System Particle Delivery MethodDNA coated microcarrier dioleoylphosphatidylethanolamine DOPE DNA DNA DNA DNA siLentFect Lipid ReagentTransFectin Lipid Reagent

3 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

4 SiRNA MLCK1 Bulletin 5370 siRNA siRNA siLentFect siRNA dsDNA vectors Suggested Reagent Quantities for Different Sizes of Plates/WellsCultureVessel SizeVolume ofPlating of 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

5 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.

6 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.

7 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, 5 147,0004 CTransFectin LipidReagent K562 DNA TransFectin LipidReagent 1well 4 105 cell 24well 24 DNA l l 1 2 3 405 Volume of TransFectin.

8 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 165-2676 Gene Pulser 10 29,0004 C165-2677 Gene Pulser 30ml 42.

9 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.

10 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