A toolkit for assembly of targeting clones for transgenesis.

Transgenic worms are a key resource for researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 GG entry vectors and 100 GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty -based GG assembly vectors that facilitate the construction of additional GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft® Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.