Abstract
Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Since the HCV genome is present exclusively in RNA form during replication, a number of anti-HCV drugs show appearance of rapid drug-resistant viruses. Therefore, it is important to test generation of drug-escape mutant viruses by developed antiviral drugs for their validity. Here, we describe a colony formation assay-based method to observe appearance of drug-resistant viruses against nucleic acid based anti-HCV drugs in genotype 1b based subgenomic replicon cell culture system (Lee et al., 2013).
Keywords: HEPATITIS C VIRUS, REPLICON, Colony forming assay
Materials and Reagents
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Cell line: HCV-replicon Huh-7 human hepatoma cell line (HCV genotype 1b subgenomic replicon pFKI389neo/NS3–3’/5.1 containing the neomycin resistant gene, provided by R. Bartenschlager, Heidelberg University, German) (Lohmann et al., 1999; Krieger et al., 2001)
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0.1% Trypsin-EDTA (WELGENE, catalog number: LS015-01 )
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Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30919.03 )
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100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, HycloneTM, catalog number: 3V30010 )
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Lipofectamine 2000 (Life Technology, catalog number: 11668-019 )
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NaCl (Sigma-Aldrich, catalog number: S5886 )
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KCl (Sigma-Aldrich, catalog number: P5405 )
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Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
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KH2PO4 (Sigma-Aldrich, catalog number: P9791 )
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NaOH (Sigma-Aldrich, catalog number: S5881 )
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1x Phosphate Buffered Saline (PBS) (see Recipes)
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Complete Dulbecco’s modified Eagle medium with high glucose (DMEM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30243.01 ) (see Recipes)
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50 mg/ml G418 (Merck KGaA, catalog number: 345810 ) (see Recipes)
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2% Paraformaldehyde solution (Sigma-Aldrich, catalog number: 158127 ) (see Recipes)
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1% Methylene blue (Duksan Scientific, catalog number: MEE0-22002 ) (see Recipes)
Equipment
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35 mm cell culture plate (Corning, catalog number: 430165 )
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100 mm cell culture plate (BD Bioscience, catalog number: 353003 )
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5% CO2, 37 °C Incubator (Thermo Fisher Scientific, catalog number: 311 )
Procedure
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Day one: HCV-replicon cells are sub-cultured and are seeded on 35 mm cell culture dish at density of 2 x 105 in 2 ml of complete DMEM.
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Day two: Before transfection, media was replaced with DMEM not containing Penicillin/Streptomycin.
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4 μM of test or control nucleic acid-based drugs were transfected using Lipofectamine 2000 reagent according to manufacturer's instruction.
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Four hours after transfection, cells were replaced with 2 ml of fresh complete DMEM containing 500 μg/ml G418, and incubated for 2 days.
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Step 2 to step 4 was repeated every 2 day during about two weeks.
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About two weeks later, cells were trypsinized and sub-cultured, and one-tenth of the cells were replated to 35 mm dishes with 2 ml of 500 μg/ml G418 containing complete DMEM.
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Step 2 to step 6 was repeated until visible colonies were obtained (usually, colonies formed one month after first transfection).
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After acquiring colonies, media was removed and cells were washed 1 time with 1ml of 1x PBS.
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Cells were treated with 1 ml of 2% paraformaldehyde for 15 min at room temperature.
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Remove the paraformaldehyde from the cells and add 1 ml of 1% methylene blue for 10 min.
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Remove the methylene blue and wash cells with 1 ml of dH2O three times.
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Cells were air-dried for 15 min, and analyze appearance of drug-resistant cells by counting the number of G418-resistant cell colonies. You can find examples of picture showing drug-resistant colonies in the Figure 4C of Reference 1 (Lee et al., 2013).
Recipes
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1x PBS
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
2 mM KH2PO4
Add dH2O to 1 L and sterilize using autoclave
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Complete DMEM medium (505 ml)
50 ml FBS
5 ml 100x Penicillin/Streptomycin solution
450 ml DMEM
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50 mg/ml G418
1 g G418 powder
Add dH2O to 20 ml and filter (0.45 μm) for sterilization
Aliquote
Store at -20 °C
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2% Paraformaldehyde solution (15 ml)
0.3 g paraformaldehyde
75 μl 10 N NaOH
Add to dH20 to 13.5 ml and incubate 60 °C until paraformaldehyde is dissolved
Add 1.5 ml of 10x PBS and store at 4 °C
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1% methylene blue (15 ml)
0.15 g methylene blue
Add to dH2O to 15 ml
Acknowledgments
This protocol was adapted from Lee et al. (2013).
Funding Source included grants from the Korea Healthcare Technology R&D Project by the Korean Ministry for Health, Welfare & Family Affairs (A100399) and National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012M3A9B6055200).
References
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Krieger, N., Lohmann, V. and Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75(10): 4614-4624.
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Lee, C. H., Lee, Y. J., Kim, J. H., Lim, J. H., Kim, J. H., Han, W., Lee, S. H., Noh, G. J. and Lee, S. W. (2013). Inhibition of hepatitis C virus (HCV) replication by specific RNA aptamers against HCV NS5B RNA replicase. J Virol 87(12): 7064-7074.
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Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. and Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285(5424): 110-113.
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Q&A
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Lee, C. H. and Lee, S. (2013). Colony Forming Assay for HCV-Replicon Cell Line.
Bio-protocol 3(24): e1009. DOI:
10.21769/BioProtoc.1009.