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.
Original language | English (US) |
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Pages (from-to) | g3.400822.2019 |
Journal | G3 (Bethesda, Md.) |
Volume | 10 |
Issue number | 2 |
DOIs | |
State | Published - Dec 17 2019 |
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Supplemental Material for Fan et al., 2020
Fan, X. (Creator), Henau, S. D. (Creator), Feinstein, J. (Creator), Miller, S. I. (Creator), Han, B. (Creator), Froekjaer Jensen, C. (Creator), Griffin, E. E. (Creator), Fan, X. (Creator), Henau, S. D. (Creator), Feinstein, J. (Creator), Miller, S. I. (Creator), Han, B. (Creator) & Griffin, E. E. (Creator), GSA Journals, 2020
DOI: 10.25387/g3.9978611.v2, http://hdl.handle.net/10754/664958
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