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Mechanistic target of rapamycin complex 1 (mTorC1) activity plays a crucial role in brain development. Here, we present an approach for rapamycin microinjection into the habenula of larval zebrafish to achieve localized inhibition of the mTorC1 pathway and explore the role of mTorC1 in habenula function. We describe steps for performing microinjections and maintaining zebrafish larvae before and after the procedure. For complete details on the use and execution of this protocol, please refer to Doszyn et al..
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11772950 | PMC |
http://dx.doi.org/10.1016/j.xpro.2024.103566 | DOI Listing |
Mol Reprod Dev
September 2025
Biology, University of Wisconsin-Oshkosh, Oshkosh, Wisconsin, USA.
Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders.
View Article and Find Full Text PDFBio Protoc
August 2025
Kenya Agricultural Livestock Research Organization, Nairobi, Kenya.
Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechnology, including viral vectors and primordial germ cell (PGC) manipulation, are limited by biosafety risks, low efficiency, and technical complexity. This protocol outlines a two-step cloning approach for generating transposon-compatible gene constructs suitable for chicken embryo microinjection.
View Article and Find Full Text PDFMethods Mol Biol
August 2025
Department of Biological Sciences, University of North Texas, Denton, TX, USA.
The movement of the Drosophila yolk protein (DmYP) across the mosquito oocyte membrane was both fortuitous and puzzling; the cells that become future offspring--oocytes--are closed off to molecules that are not specifically recognized by a receptor, but there is no obvious ortholog of the yolk protein/receptor for DmYP in mosquitoes. Nonetheless, a small fragment of DmYP was sufficient to move the massive ribonucleoprotein complex of Cas9 and a guide RNA from the open circulatory system of a female mosquito into the mosquito oocyte for targeting of the germline DNA and heritable mutation. This procedure, known as ReMOT Control, is a robust method for CRISPR/Cas9-mediated gene knockdown that has been adapted for many orders of insects, for ticks, and even for several species of crustacean by first identifying a suitable peptide for oocyte uptake, then expressing Cas9 as a fusion protein with the peptide and finally performing adult injections with expressed, purified protein and guide RNA against a gene with a visible marker phenotype.
View Article and Find Full Text PDFDev Biol
August 2025
Sorbonne Université, CNRS, Biologie Intégrative des Organismes Marins (BIOM), F-66650, France. Electronic address:
Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B.
View Article and Find Full Text PDFJ Vis Exp
July 2025
School of Biomedical Engineering, International University, Ho Chi Minh City, Vietnam; Vietnam National University, Ho Chi Minh City, Vietnam;
Microneedles have recently gained recognition as a promising method in transdermal drug delivery owing to their minimally invasive, painless nature, and they can be flexibly engineered through geometric modifications to meet specific therapeutic requirements. Hence, this study aims to develop microneedle master molds utilizing Computer Numerical Control (CNC) milling technology to create polymeric microneedles with diverse degrees of sharpness to enhance therapeutic efficacy. Previously, our team successfully optimized two critical machining parameters, feed rate, and ramp angle, while analyzing their influence on the obtained needles.
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