Transfection of Luciferase Reporter Plasmid Into PAC2 Zebrafish Cells

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We have studied the PAC-2 cell line extensively (6,7,14,15) and so have determined a set of electroporation conditions that provide optimal transfec-tion efficiency. All subsequent protocols employ these conditions.

3.4.1. Transient Transfection

1. Harvest the cells and prepare them for electroporation exactly as described in Subheading 3.3.

2. Dissolve 35 pg of plasmid DNA containing 25 pg of the luciferase reporter plasmid and 10 pg of the DNA carrier in 50 pL of water and mix with a 500-pL aliquot of resuspended cells (see Note 10).

3. Transfer each aliquot of cells mixed with DNA to a cuvet for electroporation and perform the electroporation at 0.29 KV, with capacitance set to 960 pF and resistance set to 0 Q, at room temperature using a Bio-Rad gene pulser apparatus.

4. Following electroporation dilute each reaction to 2.5 mL with L15 complete culture medium and then aliquot 250 pL per well of a 96-well fluoplate.

5. The day after transfection, remove the culture medium, wash the cell monolayer twice with 1X PBS in order to eliminate cell debris, and then add fresh medium supplemented with 0.5 mM beetle luciferin.

6. Forty-eight hours after transfection, follow the protocol described in Subheading 3.6. for monitoring luciferase activity in vivo using the TopCount NXT counter.

3.4.2. Stable Transfection

1. Harvest the cells and prepare them for electroporation exactly as described in Subheading 3.3.

2. To each 500 pL of cells, add 35 pg of plasmid DNA, containing 10 pg of carrier DNA and 25 pg of luciferase reporter (when the neo cassette is integrated into the same plasmid) or 22.5 pg of reporter and 2.5 pg of plasmid containing a neo resistance cassette (see Note 10). Also, include a negative control cell aliquot lacking plasmid DNA.

3. Perform the electroporation at 0.29 kV, 960 ^F, 0 Q. Following electroporation dilute each cell aliquot to 10 mL with L15 complete culture medium and then transfer to a 10-cm tissue-culture Petri dish to allow attachment of viable cells.

4. The day after transfection, remove the culture medium, wash the cell monolayer twice with 1X PBS in order to eliminate cell debris, and then add fresh medium.

5. Seventy-two hours following electroporation, start the selection for antibiotic resistance by supplementing the medium with the highest antibiotic concentration, 800 ^g/mL of G-418 (see Note 11).

6. Each 5 d wash the cells with 1X PBS and change the selection medium.

7. After a period of 10 to 15 d, all nonresistant cells on the negative-control plate (nontransfected cells) should have detached (see Note 11). At this stage, reduce the concentration of G-418 to 400 ^g/mL.

8. Following 1 mo of selection reduce the G-418 concentration to 250 ^g/mL (the maintenance concentration). At this stage, 100 to 200 colonies of resistant cells should be clearly visible for each electroporation reaction. Colonies typically consist of a monolayer of densely packed cells, unlike other mammalian transformed cell lines, where colonies are typically composed of multiple layers of cells.

9. It is possible to visualize G-418 resistant colonies that successfully express luciferase reporter plasmids using a simple film autoradiographic assay. Following addition of 0.5 mM beetle luciferin to the culture medium, transfer the plates of cells to a dark room, place them directly onto a sheet of X-ray film, and then expose for 8 to 12 h. Dark spots on the developed film lie below bioluminescent, luciferase-positive colonies (see Fig. 2).

10. When positive clones contain several hundred cells, they are large enough to be individually trypsinized and transferred to single wells of a 96-multiwell plate. Upon reaching confluence, transfer the cells to larger multiwell plates and then ultimately maintain them in 25-cm2 flasks. Alternatively, the clones can be trypsinized, mixed together, and subsequently propagated as a pool (see Note 12).

11. The clones should be propagated always at the maintenance concentration of G-418 and the medium changed every 5 d.

3.5. Long-Term Storage of Cells (see Note 13)

3.5.1. Freezing Cells

1. Wash a subconfluent cell monolayer twice with 1X PBS, trypsinize, and then resuspend the cells in 10 mL of L15 complete culture medium (see Subheading 3.2.).

2. Determine the density of the cell suspension by counting an aliquot with a hemocytometer and then harvest the cells by centrifugation at 200g for 5 min at 4°C and wash twice with serum-free medium.

Fig. 2. Visualizing luciferase-positive, G-418-resistant colonies. The culture medium of a Petri dish containing hundreds of G-418-resistant, stably transfected PAC-2 cell clones was supplemented with 0.5 mM luciferin and then exposed on top of an X-ray film overnight in a dark room. After developing the film, dark spots represent individual colonies positive for luciferase activity. The variation in intensity from clone to clone is probably due to differences in the number of copies of the gene inserted into the genome or their sites of integration. The markings on the film are for alignment of the plate with respect to the film.

Fig. 2. Visualizing luciferase-positive, G-418-resistant colonies. The culture medium of a Petri dish containing hundreds of G-418-resistant, stably transfected PAC-2 cell clones was supplemented with 0.5 mM luciferin and then exposed on top of an X-ray film overnight in a dark room. After developing the film, dark spots represent individual colonies positive for luciferase activity. The variation in intensity from clone to clone is probably due to differences in the number of copies of the gene inserted into the genome or their sites of integration. The markings on the film are for alignment of the plate with respect to the film.

3. Finally, resuspend the cells at a density of 2 x 106 cells/mL in freezing medium (lacking G-418). Transfer 1-mL aliquots to cryopreservation vials and place them in a -80°C freezer for 2 to 3 d.

4. Transfer frozen aliquots to liquid nitrogen for storage.

3.5.2. Thawing Cryopreserved Cells

1. Remove frozen aliquots from liquid nitrogen and place them immediately on ice for 10 to 15 min.

2. Remove the thawed cell suspension from the vial and immediately dilute it in 10 mL of complete culture medium at room temperature.

3. Centrifuge the cells at 200g for 5 min at 4°C and discard the supernatant (see Note 14).

4. Resuspend the cells in L15 complete medium (lacking G-418) and transfer them to a tissue culture flask. Typically, more than 50% of the cells should attach.

5. After 24 h change the medium for fresh L15 complete culture medium (include 250 ^g/mL G-418 for resistant clones) to remove dead, floating cells.

3.6. In Vivo Luciferase Assay

1. Seed 3 x 104 cells of the luciferase reporter clones into individual wells of a 96-well Fluoplate in 250 ^L complete culture medium containing 250 ^g/mL G-418. For transiently transfected cells, omit G-418.

2. After 12 h, replace the medium with 250 ^L complete culture medium supplemented with 0.5 mM beetle luciferin.

3. Seal the plates using TopSeal A sealing plastic and attach a bar code identifier label to the plate.

4. Load the plates into the TopCount stacker units, with each plate sandwiched between two transparent 96-well plates to allow light to access the opaque sample plates (see Note 15).

5. Program the counter to count each plate, a minimum of once each hour, and each well for a duration of 5 s in an uninterrupted cycle. Add extra empty plates if necessary to adjust the plate-counting interval.

6. Save the data from each plate as a single ASCII file for each counting cycle. Multiple files from a complete experiment can then either be imported into Microsoft Excel using the I and A import macro or alternatively, imported directly into the Chrono program for more detailed analysis (see Note 16).

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