Experiments on photochemical internalization techniques for light-directed gene delivery
Experiments on photochemical internalization techniques for light-directed gene delivery
Photochemical internalization (P C I ) allows efficient transfection by a mechanism whereby light is directed to disrupt endocytosis vesicles in cells (Berg et al. 1999). The principle of this technique is that a photosensitizer is first targeted to the membrane of the cellular endocytosis vesicles, and then light exposure causes the membrane to rupture. The endocytosed compounds (e.g. nucleic acids) are then released into the cell to act on their targets or are further translocated into the nucleus. Author: T. Friedman et al, Translator: Wei Qin et al. This experiment is from "Gene Transfer".
Operation method
Photochemical enhancement of gene transfer Move Photochemical enhancement of gene transfer material reagents Preparation of cells for transfection Appropriate cell culture medium Nucleic acids or recombinant viruses Freshly dilute nucleic acids (e.g., DNA polymers) with cell culture fluid prior to use, and freshly dilute recombinant adenovirus, adeno-associated virus, or other potentially endocytosed viruses with serum-free cell culture fluid or PBS at the infectious titer. The amount of DNA or viral vector used depends on the cell line and vector. We recommend the use of DNA at a concentration of 0.2 to 5ug/ml, with an initial viral infection titer of 5 infectious particles/cell. Phosphate Buffer Solution (PBS) Photosensitizer: AIPcSai or TPPS2a (Porphyrin products, Logan, U tah) Sodium hydroxide (N aO H ; 〇. l m 〇 l/L ) Instrumentation CO2 incubator, 37°C Light source, 670nm (AIPcS2a) or 420nm (TPPS2a): LumiSource (PCI Biotech, AS,Oslo, Norway) Cell culture dish Methods For a simple experimental plan, see Fig. 2B. 1. Inoculate the cells in a cell culture dish so that the cells are adherent to the wall. The choice of culture dish depends on the analytical method. For example, if flow cytometry is used to examine the efficiency of transgene expression in cells, a 12-well plate with 75,000 cells per well may be used. 2. Prepare a working concentration of photosensitizer and dilute it with cell culture medium before use. Use 5-20ug/m l of AIPcS2a or 0-2 ~lug/ml of TPPS2a. The photosensitizer concentration is based on the light source, light intensity and the wavelength range covered by the absorption spectrum of the photosensitizer. Be sure that all steps are performed under non-exciting light to avoid uncontrolled photosensitizer activation and to protect cells from photochemical damage. The bench light can be turned off for in vitro experiments. 3. Remove the cell culture medium. Remove the cell culture medium and add the photosensitizer-containing cell culture medium. Incubate the cells at 37°C in a CO2 incubator for 16 to 18 h. 4. Remove the photosensitizer-containing cell culture medium and wash three times with photosensitizer-free cell culture medium. Continue to incubate at 37°C for 4 h. During this period, add nucleic acids (e.g., DNA polymers) or recombinant viral vectors at specified times. Recommended incubation times are 3.5 h for non-viral gene vectors and 30 min for recombinant viral vectors. 5. Remove the vector and wash the cells again. Incubate for 30 min at 37°C in a CO2 incubator. 6- Expose the cells, using the light dose as described in the introduction. If you want to minimize the damage to the cell membrane, incubate the cells in the absence of photosensitizer for 1~4 h before exposure. 7 - Analyze the transgene efficiency after placing the cells in a dark environment and continuing to incubate for 24~48 h. For more product details, please visit Aladdin Scientific website.
Storage solution for photosensitizer: Dissolve 5 m g of AIPcS2a or 2 m g of TPPS2a in 0.2 ml of 0 -l m o l / L NaOH, add to a final volume of i ml with sterile PBS, filter for sterilization, dispense, and store at -20°C for up to 6 months. Protect the photosensitizer solution from light. Avoid prolonged storage or repeated freezing and thawing, as this may cause the photosensitizer to polymerize, resulting in a loss of effectiveness. If the photosensitizer solution does not dissolve completely, it can be treated with an ultrasonicator for a few seconds.
If the drug is transported by photochemical internalization, add both photosensitizers and macromolecules at the same time and then incubate the cells (e.g., incubate the cells for 18 h before removing the photosensitizers). However, for nucleic acid transport, it is recommended to incubate with photosensitizers first and then add nucleic acids.