Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection
Language English Country England, Great Britain Media print-electronic
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
Grant support
R01 GM078571
NIGMS NIH HHS - United States
PubMed
20692340
DOI
10.1016/j.ibmb.2010.07.012
PII: S0965-1748(10)00167-0
Knihovny.cz E-resources
- MeSH
- Bombyx genetics metabolism MeSH
- Deoxyribonucleases chemistry genetics metabolism MeSH
- DNA Breaks, Double-Stranded MeSH
- Gene Targeting methods MeSH
- Insect Proteins chemistry genetics metabolism MeSH
- RNA, Messenger genetics metabolism MeSH
- Mutagenesis * MeSH
- DNA Repair MeSH
- Zinc Fingers MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Deoxyribonucleases MeSH
- Insect Proteins MeSH
- RNA, Messenger MeSH
Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G(1) was sufficient to be used for gene targeting relying on a screen based solely on molecular methods.
References provided by Crossref.org