Henau, Sasha De
Miller, Stephanie I
Griffin, Erik E.
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Permanent link to this recordhttp://hdl.handle.net/10754/659949
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AbstractThe 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 (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, 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.
CitationFan, X., Henau, S. D., Feinstein, J., Miller, S. I., Han, B., Frøkjær-Jensen, C., & Griffin, E. E. (2019). SapTrap assembly of C. elegans MosSCI transgene vectors. doi:10.1101/805507
SponsorsWe thank Bing He and members of the Griffin lab for comments on the manuscript. We thank Katya Voronina (U. of Montana) for the plasmids pXW7.01 and pXW7.02. We thank Ann Lavanway and Zdenich Zvindrych of the Dartmouth Life Sciences Imaging Facility for assistance with imaging. The Molecular Biosciences core facility is supported by the Norris Cotton Cancer Center and by NCI grant 5P30CA023108-40. This work was supported by grants from the NIH (R01GM110194 to EEG), baseline funding from KAUST (to CFJ) and the NWO (016.Veni.181.051 to SDH). Erik Jorgensen (U. of Utah, HHMI) contributed resources to generate some of the transgenes from NIH grant R01GM095817.
PublisherCold Spring Harbor Laboratory
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