Insertion of single-copy transgenes is an important tool in C. elegansgenome engineering. Following the discovery that the Drosophilamariner element Mos1could mobilize in the C. elegansgermline (Bessereauet 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. elegansoffered increased versatility for genetic crosses and the advantage of injecting into phenotypically wild-type animals (Giordano-Santiniet al.2010, Sempleet 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-Jensenet al.2008, Frokjaer-Jensenet al.2012). The
ttTi5605(mos1)II insertion site in particular has been used for stable germline expression, and plasmids targeting
ttTi5605are also compatible with the universal mosSCI insertion sites (Frokjaer-Jensenet 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-Jensenet al.2014), however existing
ttTi5605-mosSCI plasmids use
unc-119(+)selection. Our plasmid allows use of the
ttTi5605site 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. coliOP50 as a food source.
Second, we modified the standard
unc-119selection-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 (Gibsonet 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 excisableunc-119(+)and NeoR mosSCI plasmids will be useful companions to the extensive mosSCI plasmid and strain toolkit (Frokjaer-Jensenet al.2008, Zeiser et al.2011, Frokjaer-Jensen et al.2012, Frokjaer-Jensenet al.2014).