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 of cells with resulting AAV-based targeting vectors, and isolation and verification of gene-targeted cell clones.
Keywords: Adeno-associated virus, AAV, Targeting vector, Gene targeting, Promoter trap, 2A, Internal ribosome entry site, IRES
Background
The procedures for AAV-mediated gene targeting in general (corresponding to Sections B-G of this protocol) were previously described in other protocols (Kohli et al., 2004; Rago et al., 2007; Khan et al., 2011; Howes and Schofield, 2015). However, this protocol provides a detailed description of how to perform AAV-mediated gene targeting using a 2A-based promoter–trap system for the first time.
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
This protocol was adapted from our previous work, Karnan et al. (2016). This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS; 25460395 to S. K., 15K19561 to A. O., and 25640107 to H. K.), and Takeda Science Foundation (to H. K.).
References
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