Colony Forming Assay for HCV-Replicon Cell Line   

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, Hyclone^TM, catalog number: SH30919.03 )
   
4. 100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, Hyclone^TM, 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, Hyclone^TM, 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.