Protocols

Generating Recombinant Viruses by Co-transfection

Prepare at least 10 µg of highly purified plasmid DNA for co-transfection. Impure preparations of plasmid DNA are toxic to the cells, and many cells may lyse shortly after transfection, resulting in a lower viral titer. Vector DNA purified by cesium chloride/ethidium bromide density gradient centrifugation or anion exchange chromatography (eg, QIAEX resin, QIAGEN Inc) will be sufficiently pure for co-transfection. Refer to molecular biology manuals or manufacturer's protocols for comprehensive plasmid purification techniques.

Prepare cells (one day prior to co-transfection)

1. Prepare and label three tissue culture plates: one each for the experimental co-transfection, positive co-transfection control, and negative control. Seed 2 x 10⁶ Sf9 cells onto each 60 mm tissue culture plate. Initial cell density should be 50-70% confluent. Cell attachment should be done on a flat and even surface, allowing the cells to attach firmly, usually about 10 min. Observe plates under a light microscope to check for ~ 60% confluency. If cells don't attach after that time, they are not healthy or the wrong plates have been used (eg., non-coated petri dishes). Place plates within a 25 x 150 mm culture plate and incubate overnight in 27°C humidified incubator.

Next Day

1. Experimental co-transfection: Combine 0.5 µg BD BaculoGold DNA and 2-5 µg recombinant baculovirus transfer vector, containing your insert, in a sterile microcentrifuge tube. Mix well by gentle vortexing or by flicking the tube. Quick spin tube, then let mixture sit for 5 min before adding 1 ml of Transfection Buffer B.
2. Positive control co-transfection: Combine 0.5 µg BaculoGold DNA and 2 µg pVL1392-XylE Control Transfer Vector DNA in a sterile microcentrifuge tube. Mix well by gentle vortexing or by flicking the tube. Let mixture sit for 5 min before adding 1 ml of Transfection Buffer B.
3. From the insect cell culture plates prepared the previous day for the experimental and positive control co-transfections, aspirate the old medium and replace with 1 ml of Transfection Buffer A. Make sure that the entire surface of plate is covered to prevent the cells from drying out.
4. Aspirate the old medium from the negative control plate and replace it with 5 ml fresh TNM-FH medium. Nothing else will be added to this plate.
5. Add the 1 ml of Transfection Buffer B / DNA solution, from Step 1, drop-by-drop to the experimental co-transfection plate. After every three to five drops, gently rock the plate back and forth to mix the drops with the medium. During this procedure, a fine calcium phosphate/DNA precipitate should form. This precipitate is characterized by a fine white milky color.
6. Add 1 ml of the Transfection Buffer B / XylE Positive Control DNA solution from Step 2 drop-by-drop to the positive control co-transfection control plate, as in Step 7.
7. Place each 60 x 15mm transfection plate within a 150 x 25mm culture plate. Incubate all three plates at 27ºC for 4 - 5 hours.
8. After 4 - 5 hours, remove the medium from the experimental and positive control co-transfection plates. Add 3 ml fresh TNM-FH medium and rock the plates back and forth several times. Aspirate all medium. Add 5 ml of fresh TNM-FH medium. Place each plate back within the larger 150 x 25mm plate and incubate the plates at 27ºC for 4-5 days. It is not necessary to change the media of the negative control plate.
9. After 5 days, check the three plates for signs of infection. Compare the negative and positive controls to the experimental co-transfection plate. Infected cells will appear much larger than uninfected ones, have enlarged nuclei, stop dividing, and will float in the medium. In the negative control plate (cells only), the cells should be confluent, compared to plates with cells producing virus, which will have reduced numbers of cells.
10. Collect the supernatant of the positive control and experimental co-transfection plates into sterile 15 ml conical tubes. Centrifuge out Sf9 cells at ~ 1000 RMP for 10 min. Transfer the clarified supernatant to a new sterile 15 ml conical tube labeled with the sample description and collection date. Store at 4ºC.
11. Transfected cells expressing the XylE protein can be assayed by adding 10-100 l catechol solution* to the cells after the co-transfection supernatant has been removed and replaced with fresh media. Infected cells will turn bright yellow in approximately 5 min. * Catechol solution is 500 M catechol (Sigma-Aldrich, St. Louis, MO, Cat. No. C9510) in 50 M sodium bisulfate solution (sodium bisulfate powder: Sigma-Aldrich, St. Louis, MO, Cat. No. 233714).

• Protein production can be analyzed by western blot analysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel by harvesting cells from the 100 µl well and lysing in appropriate lysing buffer.
• The virus supernatant from the 100 µl well may be kept as the first viral amplification stock, however care should be taken to avoid cross-contamination between wells containing different virus.
• To further purify the virus population, a plaque assay purification of the co-transfection supernatant may be performed using the approximate titer obtained from the EPDA.

Discussion

It may be difficult to identify infected cells as signs of infection are not always visually apparent, particularly if the transfection efficiency is low. For example, cells which are newly infected will show signs of infection (stop dividing, become enlarged and float) but may not yet be producing protein. Additionally, cells near the 5th day of infection may have begun to lyse and much of their protein may be dispersed throughout the media. To ascertain whether the initial co-transfection was successful, perform the end-point dilution assay (EPDA) or identify recombinant viruses by plaque assay. Transfection supernatants should be amplified to produce high titer virus stocks that are used for recombinant protein production. Alternatively, single recombinant viruses, obtained by plaque purification or end-point dilution assay, may be used for virus amplification. Protein expression can be analyzed by Western blot analysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel. Prepare lysate by harvesting cells from the co-transfection or use an aliquot of the supernatant (depending whether the recombinant protein is secreted or not). Spin down debris and lyse in appropriate lysing buffer. For complete protocols, please refer to Section X, or visit our website at www.bdbiosciences.com.