Author:
Date:
2016-12-20
[Abstract] Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide rather than commonly used internal ribosome entry site (IRES) in the promoter-trap system results in significantly higher AAV-mediated gene targeting efficiencies (Karnan et al., 2016). In this protocol, we describe the procedures for AAV-mediated gene targeting exploiting 2A for promoter trapping, including the construction of a targeting vector based on the platform plasmid pAAV-2Aneo or pAAV-2Aneo v2, production of AAV particles, infection ...
[摘要] 与基于质粒的靶向载体相比,基于腺相关病毒(AAV)的靶向载体具有1-4对较高的基因靶向效率。 通过将启动子捕获系统引入靶向载体中,AAV介导的基因靶向的效率进一步增加。 此外,我们发现使用核糖体跳跃2A肽而不是通常使用的内部核糖体进入位点(IRES)在启动子捕获系统中导致显着更高的AAV介导的基因靶向效率(Karnan等,2016)。 在该方案中,我们描述了AAV介导的基因靶向开发2A用于启动子捕获的程序,包括基于平台质粒pAAV-2Aneo或pAAV-2Aneo v2的靶向载体的构建,AAV颗粒的产生,细胞感染 基于AAV的靶向载体,以及基因靶向细胞克隆的分离和验证。 【背景】以前在其他方案中描述了AAV介导的基因靶向的程序(对应于本方案的BG部分)(Kohli等人,2004; Rago等人,2007; Khan等人,2011; Howes and Schofield ,2015)。 然而,该方案提供了如何使用基于2A的启动子捕获系统首次进行AAV介导的基因靶向的详细描述。
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Author:
Date:
2015-12-20
[Abstract] Hepatitis B virus (HBV) mutants can lead to vaccine failure, diagnostic failure of HBV detection, increase viral replication and resistance to antiviral agents. To study the biological characteristics of these mutations may contribute to our knowledge on viral pathogenesis. Therefore, it is essential to isolate and characterize HBV strains from patients. Here we describe the experimental methods to isolate and clone HBV DNA from patient serum. The method will facilitate isolation and functional analysis of new HBV variants.
[摘要] 乙型肝炎病毒(HBV)突变体可导致疫苗失败,HBV检测的诊断失败,增加病毒复制和对抗病毒剂的抗性。 研究这些突变的生物学特性可能有助于我们对病毒发病机制的了解。 因此,必须从患者中分离和表征HBV病毒株。 在这里我们描述从患者血清中分离和克隆HBV DNA的实验方法。 该方法将促进新的HBV变体的分离和功能分析。
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