Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx

The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori.ArticleSCIENTIFIC REPORTS. 5:15566 (2015)journal articl

Targeted mutagenesis in bombyx mori using talens

Bombyx mori is a valuable model organism of high economic importance. Its genome sequence is available, as well as basic genetic and molecular genetic tools and markers. The introduction of genome editing methods based on engineered nucleases enables precise manipulations with genomic DNA, including targeted DNA deletions, insertions, or replacements in the genome allowing gene analysis and various applications. We describe here the use of TALENs which have a simple modular design of their DNA-binding domains, are easy to prepare and proved to be efficient in targeting of a wide range of cleavage sites. Our procedure often allows the production of individuals carrying homozygous mutations as early as in the G 1 generatio

Genome Editing Advances the Structural Study of Silk

We first applied the genome edited silkworm silk (GE-silk) to interpret X-ray fiber diagram, and implied a great potential for the application of genome editing technology to the structural study of silk. The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the X-ray fiber diagram of <i>Bombyx mori</i> silkworm silk, has been widely believed but not experimentally proven to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. The above hypothesis was experimentally demonstrated from X-ray measurements of GE-silk

Pituitary Volumes and Functions in Children with Growth Hormone Deficiency: Volumetric Magnetic Resonance Findings

Purpose: The purpose of the study was to compare pituitary volumes calculated from magnetic resonance imaging (MRI) in patients with idiopathic growth hormone deficiency (GHD) without structural abnormalities in the pituitary gland with clinical and biochemical features.Methods: This study was designed as a retrospective review and the need for informed consent was waived. MRI of the head was performed for 39 male and 17 female patients ranging in age from 10 to 14 years. Pituitary volumes were calculated from sectional area and ellipsoid formula. Pituitary volumes were compared between boys and girls. Pituitary volumes were also compared with measurements of pituitary function, age, and standard deviation scores (SDS) for height.Results: Whole pituitary volumes in patients were 405.1 ± 150.6 mm3 for the male group and 348.6 ± 161.1 mm3 for the female group. No significant difference was identified between mean pituitary volumes of the female and male groups. A weak correlation was found between pituitary volume and height SDS for boys (Ï = 0.34, p = .03). For girls, a moderate correlation was identified between pituitary volume and FSH level (Ï = 0.67, p &lt; .01).Conclusion: Pituitary volumes of GHD patients correlated with height SDS in boys and FSH levels in girls. Brain MRI can be used not only to rule out anatomical abnormalities of the pituitary gland in GHD patients, but also to assess pituitary functions in clinical settings

Efficient TALEN construction for Bombyx mori gene targeting.

Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments