Year: 2019
Type: article
Abstract: The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3′UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans. Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3′UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.
Source: G3 Genes Genomes Genetics
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,709
Funders: Howard Hughes Medical Institute, Dartmouth College, Nederlandse Organisatie voor Wetenschappelijk Onderzoek, King Abdullah University of Science and Technology, National Institutes of Health, Norris Cotton Cancer Center, National Cancer Institute, National Institute of General Medical Sciences
OpenAlex ID: W2996555166