Original research article

The authors used this protocol in:
Jun 2013

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Colony Forming Assay for HCV-Replicon Cell Line    

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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

  1. 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)
  2. 0.1% Trypsin-EDTA (WELGENE, catalog number: LS015-01 )
  3. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30919.03 )
  4. 100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, HycloneTM, catalog number: 3V30010 )
  5. Lipofectamine 2000 (Life Technology, catalog number: 11668-019 )
  6. NaCl (Sigma-Aldrich, catalog number: S5886 )
  7. KCl (Sigma-Aldrich, catalog number: P5405 )
  8. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  9. KH2PO4 (Sigma-Aldrich, catalog number: P9791 )
  10. NaOH (Sigma-Aldrich, catalog number: S5881 )
  11. 1x Phosphate Buffered Saline (PBS) (see Recipes)
  12. Complete Dulbecco’s modified Eagle medium with high glucose (DMEM) (Thermo Fisher Scientific, HycloneTM, catalog number: SH30243.01 ) (see Recipes)
  13. 50 mg/ml G418 (Merck KGaA, catalog number: 345810 ) (see Recipes)
  14. 2% Paraformaldehyde solution (Sigma-Aldrich, catalog number: 158127 ) (see Recipes)
  15. 1% Methylene blue (Duksan Scientific, catalog number: MEE0-22002 ) (see Recipes)

Equipment

  1. 35 mm cell culture plate (Corning, catalog number: 430165 )
  2. 100 mm cell culture plate (BD Bioscience, catalog number: 353003 )
  3. 5% CO2, 37 °C Incubator (Thermo Fisher Scientific, catalog number: 311 )

Procedure

  1. 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.
  2. Day two: Before transfection, media was replaced with DMEM not containing Penicillin/Streptomycin.
  3. 4 μM of test or control nucleic acid-based drugs were transfected using Lipofectamine 2000 reagent according to manufacturer's instruction.
  4. Four hours after transfection, cells were replaced with 2 ml of fresh complete DMEM containing 500 μg/ml G418, and incubated for 2 days.
  5. Step 2 to step 4 was repeated every 2 day during about two weeks.
  6. 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.
  7. Step 2 to step 6 was repeated until visible colonies were obtained (usually, colonies formed one month after first transfection).
  8. After acquiring colonies, media was removed and cells were washed 1 time with 1ml of 1x PBS.
  9. Cells were treated with 1 ml of 2% paraformaldehyde for 15 min at room temperature.
  10. Remove the paraformaldehyde from the cells and add 1 ml of 1% methylene blue for 10 min.
  11. Remove the methylene blue and wash cells with 1 ml of dH2O three times.
  12. 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

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    Add dH2O to 1 L and sterilize using autoclave
  2. Complete DMEM medium (505 ml)
    50 ml FBS
    5 ml 100x Penicillin/Streptomycin solution
    450 ml DMEM
  3. 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
  4. 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
  5. 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

  1. 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. 
  2. 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.
  3. 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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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.
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