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Methods. 2014 Aug 15;69(1):38-45. doi: 10.1016/j.ymeth.2014.02.008. Epub 2014 Feb 18.

Targeted genome editing in Aedes aegypti using TALENs.

Author information

1
Fralin Life Science Institute, Dept. of Entomology, Virginia Tech, Blacksburg, VA 20461, United States.
2
Fralin Life Science Institute, Dept. of Entomology, Virginia Tech, Blacksburg, VA 20461, United States. Electronic address: zachadel@vt.edu.

Abstract

The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in A. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.

KEYWORDS:

Aedes aegypti; Gene editing; Meganuclease; Mosquito; TALE effector; TALEN

PMID:
24556554
PMCID:
PMC4136995
DOI:
10.1016/j.ymeth.2014.02.008
[Indexed for MEDLINE]
Free PMC Article

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