Format

Send to

Choose Destination
Methods. 2014 Aug 15;69(1):32-7. doi: 10.1016/j.ymeth.2014.03.020. Epub 2014 Mar 26.

A simple TALEN-based protocol for efficient genome-editing in Drosophila.

Author information

1
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
2
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: schnorrer@biochem.mpg.de.

Abstract

Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2 months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.

KEYWORDS:

Drosophila; Genome-editing; Method; Mutagenesis; T7 endonuclease I; TALEN

PMID:
24680697
DOI:
10.1016/j.ymeth.2014.03.020
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center