Protocol
The following protocol for GeneFECTOR (TM) is applicable to all of our reagents.

OPTIMIZATION OF PARAMETERS: The conditions for optimal transfection efficiency vary between different cell types. To achieve the highest possible transfection efficiencies, several parameters need to be optimized. Once these parameters have been established for a particular cell line, reasonable reproducibility can be obtain from experiment to experiment. The following are the most important parameters:

Cell Confluence: Cell confluence should be between 50-70%, and kept as constant as possible from experiment to experiment. In general, it is easier to control this parameter in medium to large wells (6 well plates, 35-60 mm culture dishes) than in smaller sizes.

DNA/Liposome Complex: The optimal ratio of DNA/liposome and the total amount of DNA/liposome complex should be determined using a constant number of cells to obtain the highest transfection efficiency. This is easily achieved starting with a constant amount of DNA (for example, 1 g per 35 mm dish) and varying the amount of GeneFECTOR (1-6 g per dish in 0.5 or 1.0 g increments). Then, keeping constant the DNA/liposome ratio which gave the highest efficiency in the previous experiment, vary the total amount of DNA/liposome complex and determine the new peak of activity. For example, if the DNA/liposome ratio in the first experiment was 1 g/1 g, then one could use 0.5 g/0.5 g, 1.0 g/1.0 g, 1.5 g/1.5 g and 2.0 g/2.0 g total amounts of DNA/liposome complex in the second experiment.

Transfection Time: Transfection efficiency is related to the exposure time of the cells to the DNA/liposome complex. In general, the longer the exposure time the higher the efficiency. However, since transfection is carried out in serum free or reduced serum medium, excessively long exposure times could lead to cell detachment or death. The recommended starting transfection time is 6-8 hour, however longer exposure times could prove optimal.

DNA TRANSFECTION OF ADHERENT CELLS (general protocol).

Cell plating: The following protocol is for 35mm dishes. For other sizes, adjust the volumes proportionally. The cells are plated in six 35mm dishes at a concentration such that their confluency will be 50-70% when transfected (usually 24-48 hours after plating).

GeneFECTOR:DNA complex: The DNA/liposome complex should be prepared at room temperature.

  • Preparation of sterile DNA for transfections: Plasmid DNA (cesium chloride or similar purity grade) is ethanol precipitated in a sterile microfuge tube, washed with 70% ethanol, and dissolved in sterile deionized water to give a final concentration of 1 g/l. Store frozen at minus 20oC.
  • Plasmid DNA (1-2 g/dish) is diluted in sterile water or serum and antibiotics free MEM to give 1 g/100l final concentration.
  • GeneFECTOR aliquots of 0, 1, 2, 4, or 6 l are diluted with serum and antibiotics free MEM to a final volume of 100 l in siliconized microfuge tubes(note1).
  • The plasmid DNA solution is added directly to the diluted liposome solutions with a pipettor and mixed by finger tapping the tubes or pipetting the liquid up and down. The DNA/liposome complex should form within seconds, however some investigators prefer to let the mixture sit for 15-45 minutes at room temperature (note 2).

Transfection:

  • Wash the cells with serum and antibiotics free MEM ( 2x 1ml). Washing with PBS is not recommended since the residual phosphate from the PBS will compete with the DNA for the liposomes.
  • Add 0.8 ml of serum and antibiotics free MEM to each well containing the cells (note 3).
  • Add the plasmid/liposome complex solutions ( 200 l) to the corresponding 35 mm wells in a dropwise fashion using a pipettor, trying to cover all areas of the well . Mix by gently swirling the plates.
  • Incubate the cells for 5-18 hours under the standard conditions used to grow the cells (normally 37 oC, 5% CO2 atmosphere). 5-7 hours is a good starting range, however longer times may be used depending upon the cell type.
  • Add 1ml of complete MEM (FBS and antibiotics) containing twice as much FBS and antibiotics as normally used to grow the cells. This attenuates the transfection and restores the serum and antibiotic concentrations to normal levels(note 4).
  • Incubate the cells for additional 18-24 hours under the usual conditions.
  • Replace the medium with complete medium.
  • Assay the cells at the appropriate time ( 24- 72 hours) and determine the concentration range for peak expression of the reporter gene product. Narrow the concentration range for optimal activity as needed.

Note 1: Unsiliconized tubes can be used, but siliconized tubes appear to give better results

Note 2: The DNA: liposome complex MUST be made in serum free medium, otherwise negatively charged macromolecules in the serum will compete with the DNA for the liposome

Note 3: If the cells require serum at all times for survival , 0.8 ml of MEM with reduced (20-50% of normal) serum, but without antibiotics, can be used instead. Antibiotics present during transfection can kill the cells and can decrease the transfection efficiency.

Note 4: For serum free human keratinocytes, no FBS addition is needed. However, rinsing with medium containing BSA (0.5%) can be done to remove excess lipid/DNA complex. Then, serum free medium is added to restore the normal culture conditions.

DNA TRANSFECTION OF SUSPENSION CELLS. The following protocol is for 35 mm culture wells or dishes, and has been shown to work well with Jurkat cells.

Preparation of cells.

  • Transfer a stock suspension cell culture to a 50 ml conical tube and centrifuge at 400 x g for 10 min.
  • Wash cells twice by aspirating the supernatant, gently resuspending the cell pellet in 10-20 ml of serum free medium, and centrifuging again at 400 x g for 10 min.
  • Resuspend the pellet in serum-free growth medium to give a final concentration of 6 x 106 cells /ml.
  • Plate six 35 mm culture wells or dishes with 0.8 ml of the cell suspension.

GeneFECTOR:DNA complex: Prepare the DNA/liposome complexes using the protocol described for adherent cells.

Transfection:

  • Transfer the DNA/liposome complex to the cultures by randomly dropping the solution over the medium.
  • Swirl the wells gently to mix and incubate at 37 0 C for 5-8 hours or longer (see discussion of exposure time above).
  • After 5-8 hour incubation, add 4 ml growth medium containing 12.5 % FBS to the wells and continue the incubation for an additional 72 hours.
  • The cells can then be transferred to 10 ml Falcon tubes, the wells rinsed with 5ml sterile PBS, and pellet at 400 x g for 10 min.
  • The cell pellet is then washed twice with 5ml sterile PBS and the final pellet resuspended in lysis buffer for assay of the reporter gene product.

* Patent pending. For transgenic use, a signed agreement is necessary.


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