Tools for Mos1-mediated single copy insertion (mosSCI) with excisable unc-119(+) or NeoR (G418) selection cassettes

Insertion of single-copy transgenes is an important tool in C. elegans genome engineering. Following the discovery that the Drosophila mariner element Mos1 could mobilize in the C. elegans germline (Bessereau et al. 2001), tools for efficient Mos1-mediated transgene insertion have been developed and widely adopted (Robert and Bessereau 2010). In parallel, the establishment of effective antibiotic selection in C. elegans offered increased versatility for genetic crosses and the advantage of injecting into phenotypically wild-type animals (Giordano-Santini et al. 2010, Semple et al. 2010). Reliable, site-specific, single-copy transgene insertion remains a key technique for applications such as synthetic reporters and rapid testing of genetic rescue constructs, especially when germline expression is required. The highly developed Mos1-mediated single-copy insertion (mosSCI) system is a toolkit of choice for this approach (Frokjaer-Jensen et al. 2008, Frokjaer-Jensen et al. 2012). The ttTi5605(mos1)II insertion site in particular has been used for stable germline expression, and plasmids targeting ttTi5605 are also compatible with the ‘universal’ mosSCI insertion sites (Frokjaer-Jensen et al. 2014). To add to the suite of tools for targeting ttTi5605, we constructed two modified mosSCI plasmids (Figure 1). First, we made a plasmid for ttTi5605-mosSCI using G418 selection. Antibiotic selection has been incorporated into the miniMos system for random transgene insertion (Frokjaer-Jensen et al. 2014), however existing ttTi5605-mosSCI plasmids use unc-119(+) selection. Our plasmid allows use of the ttTi5605 site without the need for injection into unc-119(ed3) mutants, which are more challenging to grow than wild-type animals and do not have optimal germline health on standard E. coli OP50 as a food source. Second, we modified the standard unc-119 selection-based ttTi5605-mosSCI plasmid (pCFJ151) by adding loxP recombination sites flanking the unc-119(+) selection cassette (Figure 1B). This change allows excision of the unc-119(+) selection cassette using an existing Cre recombinase protocol (Dickinson et al. 2013). Since unc-119(+) is a frequently used selection marker, this new feature will add flexibility for construction of strains with multiple transgenes. For increased versatility, both of the plasmids described here are minimal and do not encode additional tags or fluorescent proteins. Multiple-fragment transgenes can be seamlessly fused and cloned into these plasmids using the Gibson isothermal assembly technique by designing PCR amplicons, synthetic dsDNAs, restriction fragments or oligos with overlapping homologous ends (Gibson et al. 2009). We performed three independent test injections using different transgenes and found both of our plasmids to be effective for mosSCI (Figure 1). We also successfully excised the unc-119(+) cassette (Figure 1B) using Cre recombinase for two independent insertions (Methods). However, a caveat of the current NeoR plasmid (Figure 1A) is that no additional phenotypic marker was included between the loxP sites. Therefore, unlike unc-119(+) excision, which can be easily screened for using a visible phenotype, excision of NeoR would require screening by PCR or by loss of drug resistance. Overall, we hope that these excisable unc-119(+) and NeoR mosSCI plasmids will be useful companions to the extensive mosSCI plasmid and strain toolkit (Frokjaer-Jensen et al. 2008, Zeiser et al. 2011, Frokjaer-Jensen et al. 2012, Frokjaer-Jensen et al. 2014).

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