2 Max Delbrück Centrum, BIMSB, Berlin, Germany
3 Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.
4 Worm Biology Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay.
5 Rockefeller University, New York, USA
A readily available fosmid library covers ~90% of all worm genes. We have previously described a pipeline for manipulating the sequence of fosmid clones via a recombineering strategy in E.coli (Tursun et al., 2009, PLoS One. 2009;4(3):e4625). Recombineered fosmids permit expression of fluorescent reporters or genetically altered protein products, in transgenic worms, under the control of most, if not all, endogenous regulatory sequences of the gene of interest. We report here more than a dozen new cassettes for fosmid recombineering using our pipeline. These cassettes allow for a number of different types of tagging for biochemical and microscopy purposes among others, all listed in Table 1.
In light of the advent of CRISPR/Cas9-mediated genome engineering, it is important to point out the persistent value of fosmid-based transgenesis: (a) Single copy fluorescent proteins generated by CRISPR/Cas9 or miniMos can be difficult to visualize, especially if the gene has low expression. The multicopy nature of transgenic arrays can increase the intensity of the reporter gene signal to a detectable level. (b) Some genes remain difficult to target by CRISPR, either for technical or biological reasons (e.g. adding a tag generates a hypomorphic allele that might not provide enough gene function in single copy). In our hands, fosmid recombineering in bacteria is fast and always successful. Finally, (c) fosmid-based reporter transgenes provide a fast and straightforward tool for both rescue of mutant phenotypes and subsequent mosaic analysis.
The complete toolkit can be requested from the Hobert lab (email@example.com).