Liposomal vectors for direct in vivo gene transfection experiments
Liposomal vectors for direct in vivo gene transfection experiments
Many of the cationic liposomes used for liposomal transfection of cells in tissue cultures are able to be purchased. Composition optimization is highly dependent on the cell type and does not correlate with the composition that is typically optimized when performing in vivo transfection of the corresponding tissue. Therefore, for in vitro transfection, the reader is advised to test different commercial liposomes under the guidance of the instructions.
Operation method
Basic scheme Preparation of liposomes by film hydration before dosage compression molding
Materials and Instruments
Chloroform DC-Chol Storage Solution DOPE DOTAP Cholesterol Storage Solution Glucose Move 1. Rinse 30.0 ml of UV-transparent glass tubing three times with chloroform. 2. Mix the appropriate lipids in the UV-transparent glass tube for liposome preparation. (1) For DC-Chol/DOPE liposomes (molar ratio 3:2): add 1.07 ml of DC-Chol stock solution and 0.1 ml of DOPE stock solution and mix by vortexing back and forth. (2) For DOTAP/cholesterol liposomes (1:1 molar ratio): add 1.0 ml DOTAP Stock Solution and 0.55 ml Cholesterol Stock Solution and mix by vortexing back and forth. 3. Rotate the tube by hand while evaporating the chloroform by blowing nitrogen along the tube wall to form a thin lipid film. 4. Dry in a vacuum desiccator for 2~3 h to completely dry the film. To avoid loss of the lipid film under vacuum, cover the mouth of the tube with aluminum foil and poke some small holes in the top of the foil with a 26-G1/2 needle. 5. Add 2.Oml of 5.2% glucose solution to the membrane. 6. Rotate the tube to resuspend the lipids until almost all of the lipid membrane is resuspended in the solution. The lipids may remain on the walls of the tube and appear as a white solid precipitate. Several treatments in a water bath ultrasonic breaker will help dissolve the lipids into solution 7. Leave the suspension at room temperature for 2-3 h or overnight at 4°C to allow the lipids to fully hydrate. Seal with paraffin film. Heat the suspension in a water bath at 65°C for 5~10min. 8. In order to diffuse the lipid coagulum, place the lipid solution in a water bath ultrasonic breaker and sonicate until all coagulum disappears, then return to the water bath. 9. Two 1.0um polycarbonate membrane filters were placed in an extruder and the extruder was heated to 65°c for 5 min. 10. Extrude the lipid dispersion by squeezing the suspension through it 10 times. Place in a water bath after extrusion. After compression treatment of the lipid broad suspension, it should change from an opaque turbid solution to a clear and cloudy suspension. 11. Repeat steps 9-10 with 0.4um and 0.1um polycarbonate membrane filter paper to obtain small monolayers of liposomes down to 100-200 nm. store liposomes at 4°C (up to 12 months). These liposomes are ready to be mixed with DNA for gene transfection. For more product details, please visit Aladdin Scientific website.
UV-lamped glass centrifuge tubes Nitrogen tanks Vacuum dryers Water bath ultrasonic breakers Water baths Extruders Polycarbonate membrane filters