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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:
2015-05-20
[Abstract] Recent advances in DNA sequencing capacity to accurately quantify the copy number of individual variants in a large and diverse population allows in parallel determination of the phenotypic effects caused by each genetic modification. This systematic profiling approach is a combination of forward and reverse genetics, which we refer to as quantitative high-resolution genetics (qHRG). This protocol describes how to determine the relative fitness score of each variant compared to wild type (WT) virus based on its frequency determined by Illumina sequencing. Random mutagenesis techniques will be used to introduce randomization at each codon position of the targeted region, thereby generating a comprehensive input mutant library with substitutions at each position of interest (Qi et al. ...
[摘要] DNA测序能力的最近进展，准确量化大和多样群体中单个变体的拷贝数允许平行测定由每个遗传修饰引起的表型效应。这种系统分析方法是正向和反向遗传学的组合，我们称之为定量高分辨率遗传学（qHRG）。该方案描述了如何基于其通过Illumina测序确定的频率来确定每个变体与野生型（WT）病毒相比的相对适合度评分。随机诱变技术将用于在目标区域的每个密码子位置引入随机化，从而产生在每个感兴趣位置具有取代的综合输入突变文库（Qi等人，2014; Wu 等人，2014a; Wu ，2014b）。选择后，每个选定的文库将通过Illumina配对末端测序进行测序，并且确定每个突变的频率。基于频率的变化，可以用回归分析计算每个突变体的相对适合度分数。
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