Adipokinetic hormones (Akhs) are small peptides (8-10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. We employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh(1) mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene. The mutant flies were fully viable and compared to the control flies, had significantly low levels of hemolymph saccharides including trehalose and were resistant to starvation. These characteristics are similar to those obtained from the flies carrying targeted ablation of Akh-expressing neurons (reported earlier). We also found that the Akh(1) mutants are slightly heavy and had a slow metabolic rate. Furthermore, we showed that the ectopic expression of Dmel∖Akh reverses the Akh(1) phenotype and restores the wild-type characteristics. Our results confirmed that Akh is an important regulator of metabolic homeostasis in Drosophila.

• MeSH
• Drosophila melanogaster genetika   metabolismus   MeSH
• energetický metabolismus MeSH
• hemolymfa chemie   MeSH
• hmyzí hormony genetika   metabolismus   MeSH
• homeostáza MeSH
• kyselina pyrrolidonkarboxylová analogy a deriváty   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• oligopeptidy genetika   metabolismus   MeSH
• potravinová deprivace MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• tělesná hmotnost MeSH
• trehalosa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• bourec genetika   MeSH
• deoxyribonukleasy genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• mikroinjekce přístrojové vybavení   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená metody   MeSH
• oprava DNA spojením konců * MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.

• MeSH
• bourec embryologie   genetika   MeSH
• deoxyribonukleasy metabolismus   MeSH
• genetické vektory MeSH
• genový targeting metody   MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• otevřené čtecí rámce MeSH
• Saccharomyces cerevisiae MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH