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bioRxiv · 10.1101/805507

SapTrap assembly of C. elegans MosSCI transgene vectors

Fan, X.·Henau, S. D.·Feinstein, J.·Miller, S. I.·Han, B.·Frokjaer-Jensen, C.·Griffin, E. E.
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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 3UTR 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 (Schwartz and Jorgensen 2016 Genetics, 202:1277-1288). Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3UTR 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.

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Fan, X., Henau, S. D., Feinstein, J., Miller, S. I., Han, B., Frokjaer-Jensen, C., Griffin, E. E.. 2019-10-17. SapTrap assembly of C. elegans MosSCI transgene vectors. https://doi.org/10.1101/805507

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