SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

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...

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Bibliographic Details
Published in:G3 : genes - genomes - genetics 2020-02, Vol.10 (2), p.635-644
Main Authors: Fan, Xintao, De Henau, Sasha, Feinstein, Julia, Miller, Stephanie I, Han, Bingjie, Frøkjær-Jensen, Christian, Griffin, Erik E
Format: Article
Language:English
Subjects:
c. elegans
endoplasmic reticulum
Investigations
mitochondria
mossci
saptrap
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Summary: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.
ISSN:2160-1836
2160-1836
DOI:10.1534/g3.119.400822