Abstract
Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3′UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.
Highlights
Transgene silencing in the C. elegans germline has hampered research in this tissue and the early embryo
Such silencing is caused by repetitive transgene arrays that form upon injection of DNA in the gonad
The sequences that code for fluorescent proteins in the fusion constructs of the toolkit all contain syntrons, which should be advantageous for production of recombinant protein if a cDNA middle entry clone is used to generate the transgene
Summary
Transgene silencing in the C. elegans germline has hampered research in this tissue and the early embryo. Using an appropriate combination of 59 and 39 constructs with the ORF of one’s choice and one of the available destination vectors, it is easy to generate a construct that will integrate at a target site in the genome and mediate constitutive expression of an N- or C-terminal fluorescently tagged recombinant protein in the germline or early embryo.
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