Despite the prevalence of calcaneal injuries, this bone's unique properties and the progression of fracture and failure have not been adequately studied under high strain rate loading. In recent military conflicts, the incidence of UBBs has led to severe injuries, specifically in the lower extremities The foot and ankle complex, particularly the calcaneus bone, may sustain significant damage. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.Īn 'underbody blast' (UBB) is the detonation of a mine or improvised explosive device (IED) underneath a vehicle. We found that the Tg(ubb:SpCas9, u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery.

This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Recombinant Alpaca Anti-Rabbit IgG monoclonal antibody, clone WII1355 Īnti-Rabbit IgG Fc polyclonal antibody Īnti-Rabbit IgG + IgM polyclonal antibody Īnti-Rabbit IgG polyclonal antibody Recombinant Alpaca Anti-Rabbit IgG Fc monoclonal antibody, clone IB21

Cccp project virus