SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

摘要

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable 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 . 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 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.