The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
24565747
DOI
10.1016/j.ymeth.2014.02.014
PII: S1046-2023(14)00044-9
Knihovny.cz E-resources
- Keywords
- Bombyx mori, Drosophila melanogaster, Engineered nucleases, Genotyping, Golden Gate assembly, pBlue-TAL,
- MeSH
- Bombyx genetics MeSH
- Deoxyribonucleases genetics MeSH
- Drosophila melanogaster genetics MeSH
- DNA Breaks, Double-Stranded MeSH
- Microinjections instrumentation MeSH
- Molecular Sequence Data MeSH
- Mutagenesis, Site-Directed methods MeSH
- DNA End-Joining Repair * MeSH
- Base Sequence MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Deoxyribonucleases MeSH
Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.
Biology Centre of the ASCR Branisovska 31 370 05 Ceske Budejovice Czech Republic
National Institute of Agrobiological Sciences 1 2 Owashi Tsukuba Ibaraki 305 8634 Japan
References provided by Crossref.org
