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Infection of Human Hepatocyte-chimeric Mice with HBV and in vivo Treatment with εRNA
用HBV感染人肝细胞嵌合小鼠并用εRNA进行体内处理   

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Jia Li
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本实验方案简略版
Immunity
Jan 2015

Abstract

Hepatitis B virus (HBV) can cause both acute and chronic disease in human liver with potentially high risk of cirrhosis and liver cancer. The host range of non-human primates susceptible to this virus is limited. Therefore, experimental studies with human hepatocyte-chimeric mice provide an invaluable source of information regarding the biology and pathogenesis of HBV. This section describes the protocol for infection of the human hepatocyte-chimeric mice with HBV. In addition, it has recently been shown that HBV replication can be suppressed by exogenous expression of viral epsilon RNA (εRNA; Sato et al., 2015), which serves as an encapsidation signal (Bartenschlager et al., 1992). Based upon this finding, we also describe the protocol for the liposome-mediated delivery of a plasmid encoding εRNA to liver in these chimeric mice.

Keywords: Hepatitis B virus (乙型肝炎病毒), Human hepatocyte-chimeric mice (人肝细胞嵌合小鼠), Infection (感染), Epsilon RNA (εRN)

Materials and Reagents

  1. 0.1-10 μl pipet tips (Thermo Fisher Scientific, catalog number: QSP# TF104 )
  2. 1-200 μl and 100-1,000 μl pipet tips (Corning, catalog number: 4845 and 4846 , respectively)
  3. 0.2 ml 8 strips PCR tubes and caps (NIPPON Genetics, catalog number: FG-028DC )
  4. 1.5 ml and 2.0 ml microcentrifuge tubes (Corning, catalog number: MCT-150-A and MCT-200-C , respectively)
  5. 15 ml and 50 ml centrifuge tubes (Corning, catalog number: 352096 and 352070 , respectively)
  6. 96-well fast plate (NIPPON Genetics, catalog number: 38801 )
  7. 1 ml syringe (MonotaRO Co., NIPRO Genetics, catalog number: 08-010 )
  8. Human hepatocyte-chimeric mice (PhoenixBio Co.)
    Note: Chimeric mice are intravenously infected with 100 μl of HBV-C in saline solution (106 copies per mouse) derived originally from patient with chronic hepatitis (Sugiyama et al., 2006).
  9. HBV (genotype C; HBV-C) (Dr. Yasuhito Tanaka, Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Sugiyama et al., 2006)
  10. Sodium chloride (Nacalai tesque, catalog number: 31320-05 )
  11. YSK lipid (a pH-sensitive cationic lipid) (Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan) (Sato et al., 2012)
  12. Cholesterol (Avanti Polar Lipid, catalog number: 57-88-5 )
  13. 1, 2-dimyristoyl-sn-glycerol methoxypolyethyleneglycol (PEG-DMG) (NOF Corporation, catalog number: GM-020 )
  14. Nuclease free-H2O
  15. Citrate buffer (12.5 mM citrate, 500 mM NaCl)
  16. pCpGfree-mcs vector (Invivogen)
  17. Primers for vector construction
    pLKO.1 Fw SpeI: CCCACTAGTTTTCCCATGATTCCTTCATATTT
    pLKO.1 Rv BglII: CCCAGATCTAAAATTGTGGATGAATACTGCC
  18. TaqMan Universal PCR Master Mix (Life Technologies, catalog number: 4304437 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems™, catalog number: 4304437”.
  19. Primers and probe for quantification of HBV DNA from HBV-infected chimeric mice sera:
    Forward Primer: SF2: 5’-CTTCATCCTGCTGCTATGCCT-3’
    Reverse Primer: SR2: 5’-AAAGCCCAGGATGATGGGAT-3’
    Probe: SP2: FAM-ATGTTGCCCGTTTGTCCTCTAATTCCAG-TAMRA
  20. MISSION® pLKO.1-puro Empty Vector Control Plasmid DNA (Sigma-Aldrich, catalog number: SHC001 )
  21. DNA oligo nucleotides (5’-3’)
    Sense: CCGGTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTTTTG
    Antisense:
    AATTCAAAAATGTCCATGCCCCAAAGCCACCCAAGGCACAGCTTGGAGGCTTGAACAGTGGGACATGTACA
  22. Oligo nucleotides (see Recipes)

Equipment

  1. Biosafety hood in a biosafety level 3 (BSL3) facility (HITACHI, catalog number: SCV-1303 ECIIB )
  2. Pipettes (PIPETMAN P2, P20 and P1000) (Gilson Scientific, catalog number: F144801 , F123600 and F123602 , respectively)
  3. qPCR adhesive seal (NIPPON Genetics, catalog number: 4Ti-0560 )
  4. Applied Biosystems Veriti Thermal Cycler (Life Technologies, catalog number: 4375786 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems™, catalog number: 4375786”.
  5. ABI StepOnePlusTM Real-Time PCR Systems (Life Technologies, catalog number: 4379216 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems™, catalog number: 4379216”.

Procedure

  1. εRNA-MEND preparation
    1. The oligo nucleotides are annealed and inserted into an AgeI-EcoRI doubly digested pLKO.1-puro vector. With the ligated vector, the U6 promoter and εRNA-coding fragment are amplified through PCR by using a pair of primers (pLKO.1 Fw SpeI: CCCACTAGTTTTCCCATGATTCCTTCATATTT; pLKO.1 Rv BglII: CCCAGATCTAAAATTGTGGATGAATACTGCC), the PCR product is digested with SpeI and BglII and inserted into pCpGfree-mcs vector. This final construct is hereinafter called p-εRNA.


      Figure 1. A plasmid map of U6 promoter-driven εRNA expression vector (p-εRNA)

    2. The preparation of the p-εRNA loaded in a liposome carrier is based on the procedure that was previously described (Sato et al., 2012): p-εRNA or empty pCpGfree-mcs vector is formulated into lipid nanoparticles (MEND). 80 μg of p-εRNA or empty vector (in H2O) is dissolved in 120 μl of citrate buffer (12.5 mM citrate, 500 mM NaCl). Addition of this solution to 480 μl of the tertiary butanol containing YSK lipid (2,100 nmol), cholesterol (900 nmol), and 1, 2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (150 nmol) leads to spontaneous formulation of liposomal particles (εRNA-MEND or control-MEND). The prepared εRNA-MEND is stocked at 4 °C until use.

  2. Infection of human hepatocyte-chimeric mice with HBV
    Note: All procedures involving the manipulation of HBV infectious materials should be conducted within biological safety cabinets (BSL3).
    Chimeric mice are intravenously infected with 100 μl of HBV-C in saline solution (106 copies per mouse) derived originally from patient with chronic hepatitis (Sugiyama et al., 2006). Three weeks after HBV infection, the sera are prepared by collecting blood samples from the tail vein and the efficiency of infection is confirmed by measuring the number of viral genome copies in the sera of HBV-infected chimeric mice by qPCR analysis as below:
    1. Prepare HBV DNA standard sample:
      The HBV plasmid (pUC19-HBV, genotype A) is subjected to a 10-fold serial dilutions in Nuclease free-H2O ranging from 1 x 103 to 1 x 109 copies/ml, and use 10 μl of this diluted sample (ranging from 1 x 10 to 1 x 107 copies/assay) as standard to quantification of HBV DNA.
    2. Set up qPCR reaction mixtures as follows (for one sample):
      DNA samples from sera of HBV-infected chimeric mice:

      Nuclease free-H2O
      6 μl
      TaqMan Universal PCR Master Mix
      12.5 μl
      Probe SP2 (10 μM)
      0.5 μl
      Forward primer SF2 (10 μM)
      0.5 μl
      Reverse primer SR2 (10 μM)
      0.5 μl
      HBV DNA sample (from sera)
      5 μl

      25 μl

      Standard DNA samples:

      Nuclease free-H2O
      1 μl
      TaqMan Universal PCR Master Mix
      12.5 μl
      Probe SP2 (10 μM)
      0.5 μl
      Forward primer SF2 (10 μM)
      0.5 μl
      Reverse primer SR2 (10 μM)
      0.5 μl
      Standard DNA:
      10 μl
      Total
      25 μl

      Note: Use 10 μl of nuclease free-H2O instead of standard DNA as negative control. Each sample is prepared in triplicate.
    3. Start the real-time PCR following the program as below:

      Holding stage
      95 °C
      10 min

      1 cycle
      Cycling stage
      95 °C
      15 sec

      45 cycles
      60 °C
      1 min
      Data collection
      Final holding stage
      4 °C




  3. In vivo treatment with εRNA
    At 4-week postinfection, εRNA-MEND or control-MEND, is administered intravenously at a dose of 0.5 mg/kg of body weight (n=3 per group) every two days for 14 days. Serum and liver samples can be subjected to qPCR for the quantification of DNA copy numbers of HBV or other analyses.

Recipes

  1. DNA oligonucleotides used for the p-εRNA plasmid construction


    Sequence (5’→3’)
    Sense
    CCGGTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTTTTG
    Antisense
    AATTCAAAAATGTCCATGCCCCAAAGCCACCCAAGGCACAGCTTGGAGGCTTGAACAGTGGGACATGTACA

Acknowledgments

This protocol, which was adapted from Sato et al. (2015), is partially based on the earlier works by Sato et al. (2012) and Sugiyama et al. (2006). This was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research [A] [25253030] to A.T., Grant-in-Aid for Scientific Research on Innovative Areas [25115502, 23112701] to A.T., Grant-in-Aid for Young Scientists [B] [25870015] to S.S.).

References

  1. Bartenschlager, R. and Schaller, H. (1992). Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J 11(9): 3413-3420.
  2. Sato, S., Li, K., Kameyama, T., Hayashi, T., Ishida, Y., Murakami, S., Watanabe, T., Iijima, S., Sakurai, Y., Watashi, K., Tsutsumi, S., Sato, Y., Akita, H., Wakita, T., Rice, C. M., Harashima, H., Kohara, M., Tanaka, Y. and Takaoka, A. (2015). The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. Immunity 42(1): 123-132.
  3. Sato, Y., Hatakeyama, H., Sakurai, Y., Hyodo, M., Akita, H. and Harashima, H. (2012). A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. J Control Release 163(3): 267-276.
  4. Sugiyama, M., Tanaka, Y., Kato, T., Orito, E., Ito, K., Acharya, S. K., Gish, R. G., Kramvis, A., Shimada, T., Izumi, N., Kaito, M., Miyakawa, Y. and Mizokami, M. (2006). Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 44(4): 915-924.

简介

乙型肝炎病毒(HBV)可引起人类肝脏的急性和慢性疾病,具有潜在的肝硬化和肝癌的高风险。 对这种病毒敏感的非人灵长类动物的宿主范围有限。 因此,用人肝细胞嵌合小鼠的实验研究提供了关于HBV的生物学和发病机理的非常宝贵的信息来源。 本节描述了用HBV感染人肝细胞嵌合小鼠的方案。 此外,最近已经表明,HBV复制可以通过作为壳体化信号的病毒ε-RNA(εRNA; Sato等人,2015)的外源表达来抑制(Bartenschlager et al。,1992)。 基于这一发现,我们还描述了脂质体介导的在这些嵌合小鼠中将编码εRNA的质粒递送至肝的方案。

关键字:乙型肝炎病毒, 人肝细胞嵌合小鼠, 感染, εRN

材料和试剂

  1. 0.1-10μl移液管吸头(Thermo Fisher Scientific,目录号:QSP#TF104)
  2. 1-200μl和100-1000μl移液器吸头(分别为Corning,目录号:4845和4846
  3. 0.2ml 8条PCR管和盖子(NIPPON Genetics,目录号:FG-028DC
  4. 1.5ml和2.0ml微量离心管(分别为Corning,目录号:MCT-150-A和MCT-200-C >
  5. 15ml和50ml离心管(Corning,目录号:352096和352070 )。
  6. 96孔快板(NIPPON Genetics,目录号:38801)
  7. 1ml注射器(MonotaRO Co.,NIPRO Genetics,目录号:08-010)
  8. 人肝细胞嵌合小鼠(PhoenixBio Co.)
    注意:嵌合小鼠静脉内感染来源于慢性肝炎患者的100μl的在盐水溶液中的HBV-C(每只小鼠10个拷贝)(Sugiyama et al。 ,2006)。
  9. HBV(基因型C; HBV-C)(名古屋市立大学医学研究生院,日本名古屋的病毒学和肝脏单位的Yasuhito Tanaka博士; Sugiyama等人,2006) br />
  10. 氯化钠(Nacalai tesque,目录号:31320-05)
  11. YSK脂质(pH敏感性阳离子脂质)(北海道大学药学部,札幌,北海道,日本)(Sato等人,2012)
  12. 胆固醇(Avanti Polar Lipid,目录号:57-88-5)
  13. 1,2-二肉豆蔻酰-sn-甘油甲氧基聚乙二醇(PEG-DMG)(NOF Corporation,目录号:GM-020)
  14. 核酸酶游离-H 2 O 2 /
  15. 柠檬酸盐缓冲液(12.5mM柠檬酸盐,500mM NaCl)
  16. pCpGfree-mcs载体(Invivogen)
  17. 载体构建用引物
    pLKO.1 Fw SpeI:CCCACTAGTTTTCCCATGATTCCTTCATATTT
    pLKO.1 Rv BglII:CCCAGATCTAAAATTGTGGATGAATACTGCC
  18. TaqMan Universal PCR Master Mix(Life Technologies,目录号:4304437)
    注意:目前,它是"Thermo Fisher Scientific,Applied Biosystems?,目录号: 4304437 "。
  19. 用于HBV感染的嵌合小鼠血清中HBV DNA定量的引物和探针:
    正向引物:SF2:5'-CTTCATCCTGCTGCTATGCCT-3'
    反向引物:SR2:5'-AAAGCCCAGGATGATGGGAT-3'
    探针:SP2:FAM-ATGTTGCCCGTTTGTCCTCTAATTCCAG-TAMRA
  20. MISSION pLKO.1-puro空载体质粒DNA(Sigma-Aldrich,目录号:SHC001)
  21. DNA寡核苷酸(5'-3')
    检测:CCGGTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTTTTG
    反义:
    AATTCAAAAATGTCCATGCCCCAAAGCCACCCAAGGCACAGCTTGGAGGCTTGAACAGTGGGACATGTACA
  22. 寡核苷酸(参见配方)

设备

  1. 生物安全3级(BSL3)设施(HITACHI,目录号:SCV-1303 ECIIB)的生物安全罩
  2. 移液管(PIPETMAN P2,P20和P1000)(Gilson Scientific,目录号:F144801,F123600和F123602)
  3. qPCR粘合密封(NIPPON Genetics,目录号:4Ti-0560)
  4. Applied Biosystems Veriti Thermal Cycler(Life Technologies,目录号:4375786)
    注意:目前,它是"Thermo Fisher Scientific,Applied Biosystems?,目录号: 4375786 "。
  5. ABI StepOnePlusTM实时PCR系统(Life Technologies,目录号:4379216)
    注意:目前,它是"Thermo Fisher Scientific,Applied Biosystems?,目录号:

程序

  1. εRNA-MEND制剂
    1. 将寡核苷酸退火并插入AgeI-EcoRI 双重消化的pLKO.1-puro载体。用连接的载体,U6 启动子和εRNA编码片段通过PCR扩增 引物对(pLKO.1 Fw SpeI:CCCACTAGTTTTCCCATGATTCCTTCATATTT; pLKO.1 Rv BglII:CCCAGATCTAAAATTGTGGATGAATACTGCC),PCR产物为 用SpeI和BglII消化并插入到pCpGfree-mcs载体中。该最终构建体在下文中称为p-εRNA

      图1.U6启动子驱动的εRNA表达载体(p-εRNA)的质粒图

    2. 载于脂质体载体中的p-εRNA的制备是基于 对先前描述的程序(Sato et al。,2012): p-εRNA或空pCpGfree-mcs载体配制成脂质 纳米颗粒(MEND)。 80μgp-εRNA或空载体(在H 2 O中) 用120μl柠檬酸盐缓冲液(12.5mM柠檬酸盐,500mM NaCl)溶解。 将该溶液加入480μl含有叔丁醇的溶液中 YSK脂质(2,100nmol),胆固醇(900nmol) 2-二肉豆蔻酰-sn-甘油,甲氧基聚乙二醇(150nmol) ?脂质体颗粒的自发制剂(εRNA-MEND或 control-MEND)。将制备的εRNA-MEND在4℃下储存备用。

  2. 感染HBV的人肝细胞嵌合小鼠
    注意:涉及操作HBV感染性材料的所有程序都应在生物安全柜(BSL3)内进行。
    嵌合小鼠用源自慢性肝炎患者的盐水溶液(每只小鼠10个拷贝)中的100μl的HBV-C静脉内感染(Sugiyama等人,2006 )。在HBV感染三周后,通过从尾静脉收集血液样品制备血清,并通过qPCR分析测量HBV感染的嵌合小鼠的血清中病毒基因组拷贝的数目来确认感染的效率,如下:
    1. 准备HBV DNA标准样品:
      HBV质粒(pUC19-HBV, 基因型A)在核酸酶中进行10倍系列稀释 从1×10 3至1×10 9个拷贝/ml的游离-H 2 O,并使用10μl的 该稀释的样品(范围从1×10 3至1×10 7个拷贝/测定) 标准定量HBV DNA。
    2. 按如下所述设置qPCR反应混合物(对于一个样品):
      来自HBV感染的嵌合小鼠血清的DNA样品:

      核酸酶游离-H 2 O 2 / 6微升
      TaqMan通用PCR主混合物
      12.5μl
      探针SP2(10μM)
      0.5μl
      正向引物SF2(10μM)
      0.5μl
      反向引物SR2(10μM)
      0.5μl
      HBV DNA样品(来自血清)
      5微升

      25μl

      标准DNA样品:
      核酸酶游离-H 2 O 2 / 1微升
      TaqMan通用PCR主混合物
      12.5μl
      探针SP2(10μM)
      0.5μl
      正向引物SF2(10μM)
      0.5μl
      反向引物SR2(10μM)
      0.5μl
      标准DNA:
      10微升
      总计
      25μl

      注意:使用10μl的无核酸酶的-H2O代替标准DNA作为阴性对照。每个样品一式三份制备。
    3. 按照以下程序开始实时PCR:

      控制阶段
      95°C
      10分钟

      1个周期
      循环阶段
      95°C
      15秒

      45个周期
      60°C
      1 min
      数据收集
      最后等待阶段
      4°C




  3. 使用εRNA的体内治疗
    在感染后4周,以每两天0.5mg/kg体重(每组n = 3)的剂量静脉内施用εRNA-MEND或对照MEND,持续14天。血清和肝脏样品可以进行qPCR以定量HBV的DNA拷贝数或其他分析

食谱

  1. 用于p-εRNA质粒构建的DNA寡核苷酸

    序列(5'→3')
    感觉
    CCGGTGTACATGTCCCACTGTTCAAGCCTCCAAGCTGTGCCTTGGGTGGCTTTGGGGCATGGACATTTTTG
    反义
    AATTCAAAAATGTCCATGCCCCAAAGCCACCCAAGGCACAGCTTGGAGGCTTGAACAGTGGGACATGTACA

致谢

从Sato等人(2015)改编的该协议部分地基于Sato等人(2012)和Sugiyama等人的早期着作al。(2006)。这得到了来自日本教育,文化,体育,科学和技术部的资助(授予科学研究资助[A] [25253030] to AT,Grant-in-Aid for Scientific Research on Innovative Areas [25115502,23112701] AT,Grant-in-Aid for Young Scientists [B] [25870015]至SS)。

参考

  1. Bartenschlager,R。和Schaller,H。(1992)。 肝炎病毒组装由聚合酶与病毒RNA基因组中的衣壳化信号结合引发。 EMBO J 11(9):3413-3420。
  2. 佐藤, ?S.,Li,K.,Kameyama,T.,Hayashi,T.,Ishida,Y.,Murakami, Watanabe,T.,Iijima,S.,Sakurai,Y.,Watashi,K.,Tsutsumi,S.,Sato, Y.,Akita,H.,Wakita,T.,Rice,C.M.,Harashima,H.,Kohara, Tanaka,Y。和Takaoka,A。(2015)。 RNA传感器RIG-I双重作为乙型肝炎病毒的先天传感器和直接抗病毒因子。 免疫力 42(1):123-132。
  3. Sato,Y.,Hatakeyama,H.,Sakurai,Y.,Hyodo,M.,Akita,H.and Harashima,H。(2012)。 pH敏感性阳离子脂质促进脂质体siRNA的递送以及体外和体内的基因沉默活性。 163(3):267-276。
  4. Sugiyama, ?M.,Tanaka,Y.,Kato,T.,Orito,E.,Ito,K.,Acharya,S.K.,Gish, G.,Kramvis,A.,Shimada,T.,Izumi,N.,Kaito,M.,Miyakawa,Y。 Mizokami,M。(2006)。 乙型肝炎病毒基因型对病毒DNA和抗原的细胞内和细胞外表达的影响。 a> 44(4):915-924。


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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sato, S., Li, K. and Takaoka, A. (2016). Infection of Human Hepatocyte-chimeric Mice with HBV and in vivo Treatment with εRNA. Bio-protocol 6(2): e1718. DOI: 10.21769/BioProtoc.1718.
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