TY  - JOUR
TI  - Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System
AU  - Karnan, Sivasundaram 
AU  - Ota, Akinobu 
AU  - Konishi, Yuko  
AU  - Wahiduzzaman, Md 
AU  - Tsuzuki, Shinobu  
AU  - Hosokawa, Yoshitaka  
AU  - Konishi, Hiroyuki  
VL  - 6
IS  - 24
PY  - 2016
DA  - 2016/12/20
SP  - e2058
C1  - Bio-protocol 2016;6:e2058
DO  - 10.21769/BioProtoc.2058
UR  - https://doi.org/10.21769/BioProtoc.2058
AB  - 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.
KW  - Adeno-associated virus
KW  - AAV
KW  - Targeting vector
KW  - Gene targeting
KW  - Promoter trap
KW  - 2A
KW  - Internal ribosome entry site
KW  - IRES
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False