A large number of non-viral systems have been used for nucleic acid delivery to cultured cells in vitro or to specific cells in vivo. They vary considerably in terms of toxicity, immunogenicity and ability to target specific cell surface receptors or cell types. A class of linear cationic polymers containing P-cyclodextrins has shown promising results in in vivo delivery of nucleic acids, including plasmid D N A , D N A z y m e and small interfering R N A . Such polymers and nucleic acids form complexes (p 〇 lyP lex) and can be further modified with targeted ligands (e.g., transferrin) to promote preferential uptake of the complexes by cells that highly express the cognate receptor. Background knowledge of such materials and the manner in which they can be applied in vitro and in vivo is presented here.
Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
CDP for in vitro transfection Move CDP for in vitro transfection Materials reagents Complete medium (with antibiotics and serum), with cyclodextrin-containing polycation (CDP; 20mg/ml) reasonably selected for cell type. The lyophilized solid is dissolved in DNase- and RNase-free water. The reservoir solution is colorless. Nucleic Acid (80umol/L) D N A z y m e (Eurogentec) Plasmid D N A (p D N A ) Small Interference RNA (s i RNA) (D h a r m a c o n , Integrated D N A Technologies) The pDNA was amplified with bacterial DH5.0t (Invitrogen) and purified using Novagen's UltraMobius 1000 kit. DNAzyme and siRNA were chemically synthesized, purified by High Performance Liquid Chromatography (HPLC) and verified by gel electrophoresis. Nucleic acids were solubilized in DNase- and RNase-free water or RNase-free potassium acetate buffer. O p t i - M e m i low serum medium (Invitrogen) Phosphate Buffer Solution (PBS, bacteriostatic) Pancreatic enzymes Instruments Cell culture plates (6-well or 2 4-well) Cell culture box (37°C, 5 % CO2) Methods 1. The day before transfection, trypsin digest and harvest adherent cells during the exponential growth phase. Inoculate cells into 6 or 24 well plates at a density of 5x104 cells per well in 24 well plates and 2. 5x105 cells per well in 6 well plates. 2- Add I m l (24-well plates) or 5 m l (6-well plates) of complete medium per well and incubate for 24 h at 37°C, 5 % C O 2 in a cell culture incubator. 3. Prepare the complex solution: add 1x the volume of CDP storage solution (typically IOmI per well) to an equal volume of nucleic acid storage solution. For in vitro transfection, nucleic acid concentrations higher than 0.2 mg/ml will result in nucleic acids not being fully bound by an equal volume of CDP solution . In this case the mixture is usually opaque or milky and the gene expression or knockdown is poor. 4. Add 9 volumes of O p t i - M E M per volume of complex solution (180ul OptiM E M per well in a 24-well plate). 5. Aspirate complete growth medium from the wells. Wash the cells with 0.5 m l P B S . 6- Add 200ul (24-well plate) or 1000ul (6-well plate) of the complex/Opti-M E M mixture per well. The plates were placed back into the incubator for 4 h . 7. Aspirate the complex/Opti-M E M mixture from the wells. Add I m l (24-well plates) or 5 m l (6-well plates) of complete medium. 8. At the appropriate time, perform gene expression or gene knockdown assays. reagents Diamantonite-PEI (50m g/m l) A I > P E G, lactose capped (A I > P E O L a c ) AI > PEG, no ligand (AI > PEG) AI>PEG, coupled to transferrin (AEH PEO Tf ) The lyophilized solid is dissolved in water free of D N a s e and R N a s e . The storage solution is colorless (A E H P E G and A I > P E O L a c ) or red/orange (A D ^ P E O T f ) Polycationers containing cyclodextrin (CDP, 20m g/m l) The lyophilized solid is dissolved in water free of D N a s e and R N a s e . The storage solution is colorless. D-Glucose (1 0% m /V, soluble in water [D 10 W ) Glucose was dissolved in DNase- and RNase-free water and filtered through a 0.2um microporous membrane. In all in vivo experiments, the final concentration of injectable glucose solution was 5%. Mouse (20 g) Nucleic acid (80umol/L) D N A z y m e (Eurogentec) Plasmid D N A (p D N A ) Small Interference RNA (s i RNA) (D h a r m a c o n , IntegratedDNATechnologies) pDNA was amplified with bacterial DH5<x (Invitrogen) and purified using Novagen's UltraMobius 1000 kit. DNAzyme and siRNA were chemically synthesized, purified by High Performance Liquid Chromatography (HPLC) and verified by gel electrophoresis. Nucleic acids were solubilized in DNase- and RNase-free water or RNase-free potassium acetate buffer. Instrumentation 27 gauge needle I m l Syringe Methods 1- Mix CDP, AEKPEG and AD--PEO ligands in water to give a final AD : (3-C D molar ratio of I : 1. 2 . Add the above solution (about 5CVg/20 g mice) to an equal volume of nucleic acid solution. 3 . Add one volume (approx. lOOpg^Og mouse) of sterile DwW to an equal volume of the above complex solution. 4 . Aspirate the above complex solution using an Im I syringe with a 27 gauge needle. 5.Preheat the tail vein of the mouse. 6- Inject the complex solution into the tail vein within 2~3s. For more product details, please visit Aladdin Scientific website.