After cloning the gene of interest in adeno-associated ITR containing plasmid, seed three times 10 to the fifth cells into a six-well plate using pre-warmed DMEM supplemented with 10%FBS. Allow the cells to grow at 37 degrees Celsius and 5%carbon dioxide to attain 75 to 90%confluence. Next, dissolve 100 milligrams of polyethylenimine hydrochloride or PEI max in 100 milliliters of distilled water to make one microgram per microliter stock.
Adjust the pH to 7.1 using sodium hydroxide. Then sterilize the mixture using a 0.22 micrometer filter and store the reagent for one month at four degrees Celsius. In a two milliliter tube, add 1.3 microgram rep/cap plasmid, 1.3 microgram ITR containing plasmid, and 2.6 microgram adenovirus helper plasmid to serum free DMEM.
The total volume of this mixture should be 100 microliters. Prepare a negative control in a separate tube, replacing rep/cap plasmid with an unrelated plasmid. Now add 5.2 microliters of PEI max to the plasmid mixture.
This maintains an equal plasmid to PEI ratio. Gently vortex 10 to 15 times on a vortex mixer set to seven. For multiple vectors, stagger the PEI addition at one minute intervals for sufficient time in subsequent steps.
Incubate each tube for exactly 15 minutes at room temperature. Then dilute the reaction by adding 1.9 milliliters of serum free DMEM to achieve a final volume of two milliliters. Gently pipette twice to mix contents.
Aspirate media from the well. Carefully add the plasmid PEI mixture to the sides of the well to avoid cell detachment and incubate cells for 72 hours at 37 degrees Celsius and 5%carbon dioxide. Then freeze the plate of transfected cells for 30 minutes at minus 80 degrees Celsius and thaw for 30 minutes at 37 degrees Celsius.
Repeat the cycle a total of three times. In a laminar flow hood, aseptically mix each well by pipetting to disrupt cells effectively. Transfer the lysate to a two milliliter tube.
Centrifuge at 15, 000 G for 15 minutes at room temperature to remove cell debris and carefully transfer the supernatant to a new two milliliter tube and store the tube at four degrees Celsius. Next, plate the desired cell type in a 96-well plate and incubate for a target confluency of 50 to 75%depending on the transduction duration. The following day, perform a one-to-three dilution series of the crude preparation in serum free media to obtain the optimal amount of vector required for transduction.
Then aspirate the media from the 96-well plate and add 50 to 100 microliters of diluted crude preparation to the wells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide. To determine the transduction efficiency, observe the expression of the fluorescent reporter using a fluorescent microscope at 48 hours post-transduction.
For further analysis, remove the vector and wash cells once with pre-warmed PBS. Finally, terminate transduction by fixing cells in 4%paraformaldehyde for 10 minutes. Transduction efficiency data suggested AAV2 and KP1 were the most potent serotypes across all the tested cell lines.
HEPA1-6 exhibited a marked decrease in transduction compared to Huh7. AAV2 efficiently transduced undifferentiated HSKMC myoblasts and differentiated HSKMC myotubes. Different media conditions play an important role during transduction.
Mouse small intestinal organoids cultured in pre-transduction media were less effectively transduced compared to those cultured in organoid growth media.
