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Colony Forming Assay for HCV-Replicon Cell Line
丙肝病毒复制子细胞系德尔集落形成分析法   

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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(丙型肝炎病毒), REPLICON(复制子), Colony forming assay(克隆形成实验)

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. 

简介

丙型肝炎病毒(HCV)是慢性肝炎,肝硬化和肝细胞癌的主要致病因子。 由于HCV基因组在复制期间仅以RNA形式存在,许多抗HCV药物显示出快速耐药病毒的出现。 因此,重要的是通过开发的抗病毒药物来测试药物逃逸突变病毒的生成的有效性。 在这里,我们描述了基于集落形成测定的方法,观察耐药病毒对基于核酸的抗HCV药物在基于基因型1b的亚基因组复制子细胞培养系统中的外观(Lee等人,2013 )。

关键字:丙型肝炎病毒, 复制子, 克隆形成实验

材料和试剂

  1. 细胞系:HCV复制子Huh-7人肝癌细胞系(含有新霉素抗性基因的HCV基因型1b亚基因组复制子pFKI389neo/NS3-3'/5.1,由R.Bartenschlager,Heidelberg University,德国提供)(Lohmann等 al。,1999; Krieger等人,2001)
  2. 0.1%胰蛋白酶-EDTA(WELGENE,目录号:LS015-01)
  3. 胎牛血清(FBS)(Thermo Fisher Scientific,Hyclone TM ,目录号:SH30919.03)
  4. 100x青霉素/链霉素溶液(Thermo Fisher Scientific,Hyclone TM ,目录号:3V30010)
  5. Lipofectamine 2000(Life Technology,目录号:11668-019)
  6. NaCl(Sigma-Aldrich,目录号:S5886)
  7. KCl(Sigma-Aldrich,目录号:P5405)
  8. Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  9. KH sub 2 PO 4(Sigma-Aldrich,目录号:P9791)
  10. NaOH(Sigma-Aldrich,目录号:S5881)
  11. 1x磷酸盐缓冲盐水(PBS)(参见配方)
  12. 完整的具有高葡萄糖的Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Hyclone TM ,目录号:SH30243.01)(参见Recipes)
  13. 50mg/ml G418(Merck KGaA,目录号:345810)(参见Recipes)
  14. 2%多聚甲醛溶液(Sigma-Aldrich,目录号:158127)(参见Recipes)
  15. 1%亚甲蓝(Duksan Scientific,目录号:MEE0-22002)(参见配方)

设备

  1. 35mm细胞培养板(Corning,目录号:430165)
  2. 100mm细胞培养板(BD Bioscience,目录号:353003)
  3. 5%CO 2,37℃孵育器(Thermo Fisher Scientific,目录号:311)

程序

  1. 第一天:将HCV复制子细胞继代培养并以2×10 5个细胞的密度接种在2ml完全DMEM中的35mm细胞培养皿上。
  2. 第二天:在转染前,用不含青霉素/链霉素的DMEM替换培养基。
  3. 使用Lipofectamine 2000试剂根据制造商的说明转染4μM的测试或对照核酸基药物。
  4. 转染后4小时,将细胞用2ml含有500μg/ml G418的新鲜完全DMEM替换,并温育2天。
  5. 在约两周期间每2天重复步骤2至步骤4
  6. 约两周后,将细胞胰蛋白酶化并继代培养,将1/10的细胞用2ml500μg/ml含完全DMEM的G418重新置于35mm皿中。
  7. 重复步骤2至步骤6,直到获得可见菌落(通常,在首次转染后一个月形成菌落)。
  8. 获得菌落后,除去培养基,用1ml 1×PBS洗涤细胞1次
  9. 在室温下用1ml的2%多聚甲醛处理细胞15分钟。
  10. 从细胞中取出多聚甲醛,加入1ml 1%亚甲基蓝10分钟
  11. 除去亚甲蓝并用1ml dH 2 O洗涤细胞三次。
  12. 细胞空气干燥15分钟,并通过计数G418抗性细胞集落的数目分析耐药细胞的外观。 您可以在参考文献1的图4C(Lee等人,2013)中找到显示耐药菌落的图片实例。

食谱

  1. 1x PBS
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    将dH 2 O加至1L并使用高压灭菌器
    灭菌
  2. 完全DMEM培养基(505ml)
    50ml FBS
    5ml 100x青霉素/链霉素溶液 450 ml DMEM
  3. 50mg/ml G418
    1克G418粉末
    将dH 2 O加至20ml,过滤(0.45μm)灭菌
    等分
    储存于-20°C
  4. 2%多聚甲醛溶液(15ml) 0.3g多聚甲醛
    75μl10N NaOH
    加至dH 2 0至13.5ml并在60℃下孵育直至多聚甲醛溶解
    加入1.5ml的10x PBS并在4℃下保存
  5. 1%亚甲基蓝(15ml) 0.15g亚甲蓝
    加至dH 2 2至15ml

致谢

该协议改编自Lee等人(2013)。 资金来源包括韩国卫生保健技术研发项目由韩国卫生,福利和家庭事务部(A100399)和教育,科学技术部资助的韩国国家研究基金会(2012M3A9B6055200)的赠款。

参考文献

  1. Krieger,N.,Lohmann,V。和Bartenschlager,R。(2001)。 E 通过细胞培养适应性突变增强丙型肝炎病毒RNA复制。 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, 通过针对HCV NS5B RNA复制酶的特异性RNA适体抑制丙型肝炎病毒(HCV)复制。 a> J Virol 87(12):7064-7074。
  3. Lohmann,V.,Korner,F.,Koch,J.,Herian,U.,Theilmann,L.and Bartenschlager,R。(1999)。 在肝癌细胞系中复制亚基因组丙型肝炎病毒RNA。 科学 285(5424):110-113。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: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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