- Email: [email protected]
A Recombinant PPRE-Driven Luciferase Bioassay for Identification of Potential PPAR Agonists
CHAPTER FIFTEEN
A Recombinant PPRE-Driven Luciferase Bioassay for Identification of Potential PPAR Agonists Feng-Yuan Tsai, Yi-Ting Cheng, Tsui-Chun Tsou1 Laboratory of Molecular Toxicology, Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Materials 2.1 Cells and culture medium 2.2 Selection of G-418-resistant cell clones components 2.3 Luciferase assay components 3. Methods 3.1 Protocol for establishment of recombinant Huh7-PPRE-Luc cells for PPRE-driven luciferase assay 3.2 Treatments of Huh7-PPRE-Luc cells with chemicals for PPRE-driven luciferase assay 3.3 PPRE-driven luciferase assay 4. Notes Acknowledgment References
428 428 428 429 430 431 431 433 433 433 434 434
Abstract A recombinant Huh7-PPRE-Luc cell line, carrying a peroxisome proliferator response element (PPRE)-driven luciferase gene, is an efficient tool for evaluation of potential peroxisome proliferator-activated receptor (PPAR) agonists. The cells exhibited a good dose–response induction in PPRE-driven luciferase activity by three subtypes of PPAR agonists as well as by a retinoid X receptor agonist, 9-cis-retinoic acid. Here, the bioassay is fitted into a 96-well plate format for high-throughput screening purpose.
Vitamins and Hormones, Volume 94 ISSN 0083-6729 http://dx.doi.org/10.1016/B978-0-12-800095-3.00015-8
#
2014 Elsevier Inc. All rights reserved.
427
428
Feng-Yuan Tsai et al.
1. INTRODUCTION Peroxisome proliferator-activated receptor (PPAR), a member of the nuclear hormone receptor family, includes three subtypes (PPARa, PPARb/d, and PPARg) that are encoded by three different genes. PPAR functions as transcription factors regulating the expression of genes (Michalik et al., 2006). When activated by agonists, PPAR bind as heterodimers with a retinoid X receptor and bind to the peroxisome proliferator response element (PPRE) in the promoter region of those downstream target genes. The physiological responses of PPAR relate to lipid/glucose metabolism and energy homeostasis. PPAR also plays critical roles in regulating cellular differentiation and development, as well as in tumorigenesis (Belfiore, Genua, & Malaguarnera, 2009) of higher organisms (Berger & Moller, 2002; Feige, Gelman, Michalik, Desvergne, & Wahli, 2006). Therefore, identification of potential PPAR agonists would be very important to determine their roles in those PPAR-related physiological or pathological consequences. PPRE-driven reporter genes can be used to assay for the activation of PPAR by PPAR agonists. Luciferase gene is chosen as the reporter because when expressed in culture cells, it is easily analyzed with most of commercially available kits by using a microplate luminometer. Most important, establishment of a recombinant Huh7-PPRE-Luc cell line (Wang et al., 2010), carrying a stable PPRE-driven luciferase gene, does not require the routine transient transfection procedure and thus makes the analysis more convenient and reliable. For high-throughput screening purpose, we have fitted the bioassay into a white 96-well plate format using Huh7-PPRE-Luc cells. The whole procedure, from cell culture, chemical treatments, cell lysis, and finally to luciferase assay, can be carried out in one single plate.
2. MATERIALS 2.1. Cells and culture medium 1. HuH-7 cells ( Japanese Collection of Research Bioresources Cell Bank/ Health Science Research Resources Bank, Osaka, Japan). 2. Dulbecco’s modified Eagle medium (D-MEM) (high glucose, with L-glutamine) (Invitrogen): Measure out 850 mL sterile dH2O. Add
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powdered media to room-temperature sterile dH2O with gentle stirring. Rinse out inside of package to remove all traces of powder and add to media. Add 3.7 g of NaCO3 to media. Add dH2O to 900 mL. Stir for 1 h until dissolved. Adjust pH of media to 7.3 using 1 N NaOH or 1 N HCl (add slowly with stirring). Sterilize immediately by using a 0.22 mm vacuum filter top (Millipore). Store media at 4 C in the dark for up to 12 months. 3. Complete D-MEM: D-MEM containing 10% fetal bovine serum (FBS) and antibiotics (100 units penicillin and 100 mg streptomycin/mL) (Invitrogen).
2.2. Selection of G-418-resistant cell clones components 1. PPRE 3-tk-Luc plasmid is a reporter construct containing three copies of PPRE upstream of a thymidine kinase promoter fused to a luciferase gene (Kliewer, Umesono, Noonan, Heyman, & Evans, 1992). The plasmid was kindly provided by Dr Ronald M. Evans (Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA, United States) (Fig. 15.1A). 2. pSUPER-EGFP-neo (Oligoengine), carrying enhanced green fluorescence protein (EGFP) and neo genes, is a selection plasmid (Fig. 15.1B). 3. Lipofectamine™ 2000 Transfection Reagent (Invitrogen).
Figure 15.1 Two plasmid constructs for establishing the recombinant Huh7-PPRE-Luc cells. (A) PPRE 3-tk-Luc plasmid is a reporter construct containing three copies of PPRE (PPRE 3) upstream of a thymidine kinase (tk) promoter fused to a luciferase gene. (B) pSUPER-EGFP-neo, carrying EGFP and neo genes, is a selection plasmid.
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4. Preparation of the DNA–Lipofectamine™ 2000 complexes. a. Dilute PPRE 3-tk-Luc plasmid DNA (2.5 mg) and pSUPEREGFP-neo plasmid DNA (0.5 mg) in 250 mL of D-MEM without serum. Mix gently (see Note 1). b. Mix Lipofectamine™ 2000 gently before use and then dilute 5 mL of Lipofectamine™ 2000 in 250 mL of D-MEM without serum. Mix gently (see Note 2). c. Combine the diluted DNA with the diluted Lipofectamine™ 2000 (total volume is 500 mL). Mix gently and incubate for 1 h at room temperature to allow the DNA–Lipofectamine™ 2000 complexes to form (see Note 3). 5. High-selection D-MEM: Complete D-MEM containing 800 mg G418/mL (Infinigen Biotech Inc.). Median-selection D-MEM: Complete D-MEM containing 200 mg G418/mL. Low-selection D-MEM: Complete D-MEM containing 50 mg G418/mL. 6. Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Invitrogen): Measure out 1000 mL sterile dH2O. Add D-PBS powder to room-temperature sterile dH2O with gentle stirring. Rinse out inside of package to remove all traces of powder and add to the buffered solution. Stir for 1 h until dissolved. Sterilize immediately by autoclaving. The final D-PBS contains 2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, and 1.15 g/L Na2HPO4. 7. Cloning cylinder (6 8 mm) (BELLCO). 8. Trypsin-EDTA (10 ) (Invitrogen): Contains 5.0 g/L trypsin, 2.0 g/L EDTA4Na, and 8.5 g/L NaCl, but no phenol red. Aseptically prepare 1 Trypsin-EDTA in D-PBS (see Note 4).
2.3. Luciferase assay components 1. Nunc® FluoroNunc™ 96-well white plate (Nalge Nunc). 2. Luciferase Assay System (Promega). 3. 1 Reporter Lysis Buffer: Add 4 v of water to 1 v of 5 Reporter Lysis Buffer (Promega). Equilibrate 1 Reporter Lysis Buffer to room temperature before use. 4. Incubator/shaker (Labnet VorTemp 56 EVC, Labnet International, Inc.). 5. Programmed microplate luminometer (MicroLumatPlus LB96V, EG&G Berthold).
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3. METHODS 3.1. Protocol for establishment of recombinant Huh7PPRE-Luc cells for PPRE-driven luciferase assay 1. HuH-7 cells are routinely maintained in complete D-MEM in a humidified atmosphere containing 5% CO2 at 37 C. 2. One day before transfection, plate 6 105 HuH-7 cells in complete D-MEM per 60 mm dish so that the cells will be 90–95% at the time of transfection. 3. Before transfection, the HuH-7 cells were washed with 2 mL of D-MEM (without serum and antibiotics) twice. The cells are cotransfected with a reporter vector, PPRE 3-tk-Luc, and a selection vector, pSUPER-EGFP-neo, using the Lipofectamine™ 2000 Transfection Reagent (Invitrogen) according to the provided protocol with modifications. 4. Add 500 mL of DNA–Lipofectamine™ 2000 complexes (see Section 2.2) to each dish containing cells and then add another 1 mL of D-MEM (without serum and antibiotics) (total volume is 1.5 mL). Mix gently by rocking the dishes back and forth. 5. Incubate the cells at 37 C in a CO2 incubator for 6 h, add another 1.5 mL of D-MEM (with 20% FBS and without antibiotics) (total volume is 3 mL), and then incubate the cells at 37 C in a CO2 incubator overnight. 6. Maintain the transfected cells in 2 mL of high-selection D-MEM for 6 days (replace with fresh selection medium every 3 days) and then in median-selection D-MEM for another 4 days. 7. Following the 10-day G418 selection, scrape those G418-resistant/ EGFP-negative cell colonies and keep those G418-resistant/EGFPpositive cell colonies. Maintain the G418-resistant/EGFP-positive cell colonies in 2 mL of median-selection D-MEM for 4 days. 8. After the G418 selection, examine those well-isolated cell clones with G418-resistant/EGFP-positive phenotype with an inverted microscope. 9. Draw a circle around the selected colonies on the bottom of the dish with a marking pen. 10. Remove and discard the growth medium. Rinse the dish with D-PBS.
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11. Using sterile medium forceps, pick up a cloning cylinder (6 8 mm). Gently press the flat bottom of the cylinder into the smooth silicone grease and remove with a sudden vertical motion. If done properly, this will give even distribution of grease on the bottom of the cylinder. Set the cylinder over a colony. Gently press the cylinder down evenly with the forceps (see Note 5). 12. Add about 0.1 mL of the 1 trypsin–EDTA to the cloning cylinder and then incubate the dish at 37 C for 5 min. 13. Add a few drops of growth medium to the cylinder and gently aspirate the cells with a micropipettor. 14. Transfer each cell colony to a well in a 24-well dish with 1 mL of complete D-MEM and then incubate the cells in a humidified atmosphere containing 5% CO2 at 37 C overnight (see Note 6). 15. Maintain the cells in 1 mL of low-selection D-MEM and replace with fresh medium every 3 days till the cells grow to confluence (in about 1 week).
Figure 15.2 Dose–response induction in PPRE-driven luciferase activity by PPAR agonists. Huh7-PPRE-Luc cells were left untreated or treated with (A) WY-14643 (a PPARa agonist), (B) L-165,041 (a PPARb/d agonist), (C) Fmoc-Leu (a PPARg agonist), and (D) troglitazone (a PPARg agonist) for 24 h. Relative PPRE-driven luciferase activity is expressed as induction of PPRE-driven luciferase activity (fold) as compared with those of untreated controls. All data are presented as means SD (n ¼ 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the untreated controls. Reproduced from Wang et al. (2010) with permission from Springer.
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16. Amplify the cells in low-selection D-MEM and test the cells with the PPRE-driven luciferase activation by different PPAR agonists. As previously described, a Huh7-PPRE-Luc cell clone was established for its sharp dose–response in luciferase induction by PPAR agonists (Fig. 15.2; Wang et al., 2010).
3.2. Treatments of Huh7-PPRE-Luc cells with chemicals for PPRE-driven luciferase assay 1. Routinely maintain Huh7-PPRE-Luc cells in low-selection D-MEM. 2. One day before analysis, plate Huh7-PPRE-Luc cells at a density of 1 104 of cells per well on a Nunc® FluoroNunc™ 96-well plate (white) with complete D-MEM medium. 3. For PPRE-driven luciferase assay, replace the culture medium with 90 mL of D-MEM (without FBS), add 10 mL of treated chemicals (10) per well, and then incubate the cells for 24 h (see Note 7).
3.3. PPRE-driven luciferase assay 1. Analyze PPRE-driven luciferase activity using a Luciferase Assay System (Promega) (see Note 8). 2. After treatments, carefully remove as much the growth medium as possible from cells to be assayed (see Note 9), directly add 50 mL of 1 Reporter Lysis Buffer per well, and then shake the plate in an incubator/shaker (Labnet VorTemp 56 EVC, Labnet International, Inc.) at 900 rpm and 37 C for 10 min. 3. Program the luminometer for the delay time (2 s) and measurement time (10 s). 4. Place the plate, containing 50 mL of cell lysate per well, into the luminometer with injector. The injector adds 100 mL of Luciferase Assay Reagent per well and then the well is read immediately. The plate is advanced to the next well for a repeat of the inject-then-read process. 5. Measure the light produced for a period of 10 s (see Note 10). Luciferase activity was expressed as relative light units/well.
4. NOTES 1. To eliminate the possibility of spontaneous G418-resistant cell clone, selection of the cell clones was based on two criteria, that is, G418-resistant and EGFP-positive, contributed by pSUPER-EGFP-neo. To increase the probability that the selected G418-resistant/EGFP-positive cell clones
434
2. 3. 4. 5. 6. 7.
8. 9. 10.
Feng-Yuan Tsai et al.
also carried the PPRE 3-tk-Luc plasmid DNA, we performed transfection using the DNA mixture of 2.5 mg of PPRE 3-tk-Luc and 0.5 mg of pSUPER-EGFP-neo (PPRE 3-tk-Luc/pSUPER-EGFP-neo ¼ 5). Combine the diluted Lipofectamine™ 2000 with the diluted DNA within 5 min. Longer incubation times may decrease activity. DNA–Lipofectamine™ 2000 complexes are stable for 6 h at room temperature. Both Ca2þ and Mg2þ inhibit trypsin activity. Make sure to prepare 1 Trypsin-EDTA in balanced salt solution without Ca2þ and Mg2þ. Uneven pressure will cause the grease seal to leak. Be very careful not to slide the cylinder across the colony. This will smear the silicone grease over the cells and prevent the trypsin from contacting them. Smaller colonies or diluting the cells too much will often result in slow or no growth. The chemical-induced cytotoxicity may result in decreases in luciferase activity. For those chemicals dissolved in DMSO, dilute the stock chemicals to 10 of final concentrations with D-MEM (without FBS). Final DMSO concentration in culture medium has to be 1%, otherwise results in cytotoxicity. The Luciferase Assay Reagent and samples should be at ambient temperature prior to performing a luciferase assay. Residual growth medium left in the well may decrease the luciferase activity. The light intensity of the reaction is nearly constant for about 1 min and then decays slowly, with a half-life of approximately 10 min. The typical delay time is 2 s, and the typical read time is 10 s. The assay time may be shortened significantly to decrease the total read time if sufficient light is produced.
ACKNOWLEDGMENT This work was supported by grants from National Health Research Institutes in Taiwan.
REFERENCES Belfiore, A., Genua, M., & Malaguarnera, R. (2009). PPAR-g agonists and their effects on IGF-I receptor signaling: Implications for cancer. PPAR Research, 2009. Article ID 830501, 18 pages. http://dx.doi.org/10.1155/2009/830501. Berger, J., & Moller, D. E. (2002). The mechanisms of action of PPARs. Annual Review of Medicine, 53, 409–435. Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: Peroxisome proliferator-activated receptors are nuclear
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receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45, 120–159. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., & Evans, R. M. (1992). Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature, 358, 771–774. Michalik, L., Auwerx, J., Berger, J. P., Chatterjee, V. K., Glass, C. K., Gonzalez, F. J., et al. (2006). International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors. Pharmacological Reviews, 58, 726–741. Wang, Y. F., Chao, H. R., Wu, C. H., Tseng, C. H., Kuo, Y.-T., & Tsou, T.-C. (2010). A recombinant peroxisome proliferator response element-driven luciferase assay for evaluation of potential environmental obesogens. Biotechnology Letters, 32, 1789–1796.
A Recombinant PPRE-Driven Luciferase Bioassay for Identification of Potential PPAR Agonists Feng-Yuan Tsai, Yi-Ting Cheng, Tsui-Chun Tsou1 Laboratory of Molecular Toxicology, Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Materials 2.1 Cells and culture medium 2.2 Selection of G-418-resistant cell clones components 2.3 Luciferase assay components 3. Methods 3.1 Protocol for establishment of recombinant Huh7-PPRE-Luc cells for PPRE-driven luciferase assay 3.2 Treatments of Huh7-PPRE-Luc cells with chemicals for PPRE-driven luciferase assay 3.3 PPRE-driven luciferase assay 4. Notes Acknowledgment References
428 428 428 429 430 431 431 433 433 433 434 434
Abstract A recombinant Huh7-PPRE-Luc cell line, carrying a peroxisome proliferator response element (PPRE)-driven luciferase gene, is an efficient tool for evaluation of potential peroxisome proliferator-activated receptor (PPAR) agonists. The cells exhibited a good dose–response induction in PPRE-driven luciferase activity by three subtypes of PPAR agonists as well as by a retinoid X receptor agonist, 9-cis-retinoic acid. Here, the bioassay is fitted into a 96-well plate format for high-throughput screening purpose.
Vitamins and Hormones, Volume 94 ISSN 0083-6729 http://dx.doi.org/10.1016/B978-0-12-800095-3.00015-8
#
2014 Elsevier Inc. All rights reserved.
427
428
Feng-Yuan Tsai et al.
1. INTRODUCTION Peroxisome proliferator-activated receptor (PPAR), a member of the nuclear hormone receptor family, includes three subtypes (PPARa, PPARb/d, and PPARg) that are encoded by three different genes. PPAR functions as transcription factors regulating the expression of genes (Michalik et al., 2006). When activated by agonists, PPAR bind as heterodimers with a retinoid X receptor and bind to the peroxisome proliferator response element (PPRE) in the promoter region of those downstream target genes. The physiological responses of PPAR relate to lipid/glucose metabolism and energy homeostasis. PPAR also plays critical roles in regulating cellular differentiation and development, as well as in tumorigenesis (Belfiore, Genua, & Malaguarnera, 2009) of higher organisms (Berger & Moller, 2002; Feige, Gelman, Michalik, Desvergne, & Wahli, 2006). Therefore, identification of potential PPAR agonists would be very important to determine their roles in those PPAR-related physiological or pathological consequences. PPRE-driven reporter genes can be used to assay for the activation of PPAR by PPAR agonists. Luciferase gene is chosen as the reporter because when expressed in culture cells, it is easily analyzed with most of commercially available kits by using a microplate luminometer. Most important, establishment of a recombinant Huh7-PPRE-Luc cell line (Wang et al., 2010), carrying a stable PPRE-driven luciferase gene, does not require the routine transient transfection procedure and thus makes the analysis more convenient and reliable. For high-throughput screening purpose, we have fitted the bioassay into a white 96-well plate format using Huh7-PPRE-Luc cells. The whole procedure, from cell culture, chemical treatments, cell lysis, and finally to luciferase assay, can be carried out in one single plate.
2. MATERIALS 2.1. Cells and culture medium 1. HuH-7 cells ( Japanese Collection of Research Bioresources Cell Bank/ Health Science Research Resources Bank, Osaka, Japan). 2. Dulbecco’s modified Eagle medium (D-MEM) (high glucose, with L-glutamine) (Invitrogen): Measure out 850 mL sterile dH2O. Add
High-Throughput Screening for PPAR Chemicals
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powdered media to room-temperature sterile dH2O with gentle stirring. Rinse out inside of package to remove all traces of powder and add to media. Add 3.7 g of NaCO3 to media. Add dH2O to 900 mL. Stir for 1 h until dissolved. Adjust pH of media to 7.3 using 1 N NaOH or 1 N HCl (add slowly with stirring). Sterilize immediately by using a 0.22 mm vacuum filter top (Millipore). Store media at 4 C in the dark for up to 12 months. 3. Complete D-MEM: D-MEM containing 10% fetal bovine serum (FBS) and antibiotics (100 units penicillin and 100 mg streptomycin/mL) (Invitrogen).
2.2. Selection of G-418-resistant cell clones components 1. PPRE 3-tk-Luc plasmid is a reporter construct containing three copies of PPRE upstream of a thymidine kinase promoter fused to a luciferase gene (Kliewer, Umesono, Noonan, Heyman, & Evans, 1992). The plasmid was kindly provided by Dr Ronald M. Evans (Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, CA, United States) (Fig. 15.1A). 2. pSUPER-EGFP-neo (Oligoengine), carrying enhanced green fluorescence protein (EGFP) and neo genes, is a selection plasmid (Fig. 15.1B). 3. Lipofectamine™ 2000 Transfection Reagent (Invitrogen).
Figure 15.1 Two plasmid constructs for establishing the recombinant Huh7-PPRE-Luc cells. (A) PPRE 3-tk-Luc plasmid is a reporter construct containing three copies of PPRE (PPRE 3) upstream of a thymidine kinase (tk) promoter fused to a luciferase gene. (B) pSUPER-EGFP-neo, carrying EGFP and neo genes, is a selection plasmid.
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Feng-Yuan Tsai et al.
4. Preparation of the DNA–Lipofectamine™ 2000 complexes. a. Dilute PPRE 3-tk-Luc plasmid DNA (2.5 mg) and pSUPEREGFP-neo plasmid DNA (0.5 mg) in 250 mL of D-MEM without serum. Mix gently (see Note 1). b. Mix Lipofectamine™ 2000 gently before use and then dilute 5 mL of Lipofectamine™ 2000 in 250 mL of D-MEM without serum. Mix gently (see Note 2). c. Combine the diluted DNA with the diluted Lipofectamine™ 2000 (total volume is 500 mL). Mix gently and incubate for 1 h at room temperature to allow the DNA–Lipofectamine™ 2000 complexes to form (see Note 3). 5. High-selection D-MEM: Complete D-MEM containing 800 mg G418/mL (Infinigen Biotech Inc.). Median-selection D-MEM: Complete D-MEM containing 200 mg G418/mL. Low-selection D-MEM: Complete D-MEM containing 50 mg G418/mL. 6. Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Invitrogen): Measure out 1000 mL sterile dH2O. Add D-PBS powder to room-temperature sterile dH2O with gentle stirring. Rinse out inside of package to remove all traces of powder and add to the buffered solution. Stir for 1 h until dissolved. Sterilize immediately by autoclaving. The final D-PBS contains 2 g/L KCl, 0.2 g/L KH2PO4, 8 g/L NaCl, and 1.15 g/L Na2HPO4. 7. Cloning cylinder (6 8 mm) (BELLCO). 8. Trypsin-EDTA (10 ) (Invitrogen): Contains 5.0 g/L trypsin, 2.0 g/L EDTA4Na, and 8.5 g/L NaCl, but no phenol red. Aseptically prepare 1 Trypsin-EDTA in D-PBS (see Note 4).
2.3. Luciferase assay components 1. Nunc® FluoroNunc™ 96-well white plate (Nalge Nunc). 2. Luciferase Assay System (Promega). 3. 1 Reporter Lysis Buffer: Add 4 v of water to 1 v of 5 Reporter Lysis Buffer (Promega). Equilibrate 1 Reporter Lysis Buffer to room temperature before use. 4. Incubator/shaker (Labnet VorTemp 56 EVC, Labnet International, Inc.). 5. Programmed microplate luminometer (MicroLumatPlus LB96V, EG&G Berthold).
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3. METHODS 3.1. Protocol for establishment of recombinant Huh7PPRE-Luc cells for PPRE-driven luciferase assay 1. HuH-7 cells are routinely maintained in complete D-MEM in a humidified atmosphere containing 5% CO2 at 37 C. 2. One day before transfection, plate 6 105 HuH-7 cells in complete D-MEM per 60 mm dish so that the cells will be 90–95% at the time of transfection. 3. Before transfection, the HuH-7 cells were washed with 2 mL of D-MEM (without serum and antibiotics) twice. The cells are cotransfected with a reporter vector, PPRE 3-tk-Luc, and a selection vector, pSUPER-EGFP-neo, using the Lipofectamine™ 2000 Transfection Reagent (Invitrogen) according to the provided protocol with modifications. 4. Add 500 mL of DNA–Lipofectamine™ 2000 complexes (see Section 2.2) to each dish containing cells and then add another 1 mL of D-MEM (without serum and antibiotics) (total volume is 1.5 mL). Mix gently by rocking the dishes back and forth. 5. Incubate the cells at 37 C in a CO2 incubator for 6 h, add another 1.5 mL of D-MEM (with 20% FBS and without antibiotics) (total volume is 3 mL), and then incubate the cells at 37 C in a CO2 incubator overnight. 6. Maintain the transfected cells in 2 mL of high-selection D-MEM for 6 days (replace with fresh selection medium every 3 days) and then in median-selection D-MEM for another 4 days. 7. Following the 10-day G418 selection, scrape those G418-resistant/ EGFP-negative cell colonies and keep those G418-resistant/EGFPpositive cell colonies. Maintain the G418-resistant/EGFP-positive cell colonies in 2 mL of median-selection D-MEM for 4 days. 8. After the G418 selection, examine those well-isolated cell clones with G418-resistant/EGFP-positive phenotype with an inverted microscope. 9. Draw a circle around the selected colonies on the bottom of the dish with a marking pen. 10. Remove and discard the growth medium. Rinse the dish with D-PBS.
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11. Using sterile medium forceps, pick up a cloning cylinder (6 8 mm). Gently press the flat bottom of the cylinder into the smooth silicone grease and remove with a sudden vertical motion. If done properly, this will give even distribution of grease on the bottom of the cylinder. Set the cylinder over a colony. Gently press the cylinder down evenly with the forceps (see Note 5). 12. Add about 0.1 mL of the 1 trypsin–EDTA to the cloning cylinder and then incubate the dish at 37 C for 5 min. 13. Add a few drops of growth medium to the cylinder and gently aspirate the cells with a micropipettor. 14. Transfer each cell colony to a well in a 24-well dish with 1 mL of complete D-MEM and then incubate the cells in a humidified atmosphere containing 5% CO2 at 37 C overnight (see Note 6). 15. Maintain the cells in 1 mL of low-selection D-MEM and replace with fresh medium every 3 days till the cells grow to confluence (in about 1 week).
Figure 15.2 Dose–response induction in PPRE-driven luciferase activity by PPAR agonists. Huh7-PPRE-Luc cells were left untreated or treated with (A) WY-14643 (a PPARa agonist), (B) L-165,041 (a PPARb/d agonist), (C) Fmoc-Leu (a PPARg agonist), and (D) troglitazone (a PPARg agonist) for 24 h. Relative PPRE-driven luciferase activity is expressed as induction of PPRE-driven luciferase activity (fold) as compared with those of untreated controls. All data are presented as means SD (n ¼ 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the untreated controls. Reproduced from Wang et al. (2010) with permission from Springer.
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16. Amplify the cells in low-selection D-MEM and test the cells with the PPRE-driven luciferase activation by different PPAR agonists. As previously described, a Huh7-PPRE-Luc cell clone was established for its sharp dose–response in luciferase induction by PPAR agonists (Fig. 15.2; Wang et al., 2010).
3.2. Treatments of Huh7-PPRE-Luc cells with chemicals for PPRE-driven luciferase assay 1. Routinely maintain Huh7-PPRE-Luc cells in low-selection D-MEM. 2. One day before analysis, plate Huh7-PPRE-Luc cells at a density of 1 104 of cells per well on a Nunc® FluoroNunc™ 96-well plate (white) with complete D-MEM medium. 3. For PPRE-driven luciferase assay, replace the culture medium with 90 mL of D-MEM (without FBS), add 10 mL of treated chemicals (10) per well, and then incubate the cells for 24 h (see Note 7).
3.3. PPRE-driven luciferase assay 1. Analyze PPRE-driven luciferase activity using a Luciferase Assay System (Promega) (see Note 8). 2. After treatments, carefully remove as much the growth medium as possible from cells to be assayed (see Note 9), directly add 50 mL of 1 Reporter Lysis Buffer per well, and then shake the plate in an incubator/shaker (Labnet VorTemp 56 EVC, Labnet International, Inc.) at 900 rpm and 37 C for 10 min. 3. Program the luminometer for the delay time (2 s) and measurement time (10 s). 4. Place the plate, containing 50 mL of cell lysate per well, into the luminometer with injector. The injector adds 100 mL of Luciferase Assay Reagent per well and then the well is read immediately. The plate is advanced to the next well for a repeat of the inject-then-read process. 5. Measure the light produced for a period of 10 s (see Note 10). Luciferase activity was expressed as relative light units/well.
4. NOTES 1. To eliminate the possibility of spontaneous G418-resistant cell clone, selection of the cell clones was based on two criteria, that is, G418-resistant and EGFP-positive, contributed by pSUPER-EGFP-neo. To increase the probability that the selected G418-resistant/EGFP-positive cell clones
434
2. 3. 4. 5. 6. 7.
8. 9. 10.
Feng-Yuan Tsai et al.
also carried the PPRE 3-tk-Luc plasmid DNA, we performed transfection using the DNA mixture of 2.5 mg of PPRE 3-tk-Luc and 0.5 mg of pSUPER-EGFP-neo (PPRE 3-tk-Luc/pSUPER-EGFP-neo ¼ 5). Combine the diluted Lipofectamine™ 2000 with the diluted DNA within 5 min. Longer incubation times may decrease activity. DNA–Lipofectamine™ 2000 complexes are stable for 6 h at room temperature. Both Ca2þ and Mg2þ inhibit trypsin activity. Make sure to prepare 1 Trypsin-EDTA in balanced salt solution without Ca2þ and Mg2þ. Uneven pressure will cause the grease seal to leak. Be very careful not to slide the cylinder across the colony. This will smear the silicone grease over the cells and prevent the trypsin from contacting them. Smaller colonies or diluting the cells too much will often result in slow or no growth. The chemical-induced cytotoxicity may result in decreases in luciferase activity. For those chemicals dissolved in DMSO, dilute the stock chemicals to 10 of final concentrations with D-MEM (without FBS). Final DMSO concentration in culture medium has to be 1%, otherwise results in cytotoxicity. The Luciferase Assay Reagent and samples should be at ambient temperature prior to performing a luciferase assay. Residual growth medium left in the well may decrease the luciferase activity. The light intensity of the reaction is nearly constant for about 1 min and then decays slowly, with a half-life of approximately 10 min. The typical delay time is 2 s, and the typical read time is 10 s. The assay time may be shortened significantly to decrease the total read time if sufficient light is produced.
ACKNOWLEDGMENT This work was supported by grants from National Health Research Institutes in Taiwan.
REFERENCES Belfiore, A., Genua, M., & Malaguarnera, R. (2009). PPAR-g agonists and their effects on IGF-I receptor signaling: Implications for cancer. PPAR Research, 2009. Article ID 830501, 18 pages. http://dx.doi.org/10.1155/2009/830501. Berger, J., & Moller, D. E. (2002). The mechanisms of action of PPARs. Annual Review of Medicine, 53, 409–435. Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: Peroxisome proliferator-activated receptors are nuclear
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