A universal Cbr-unc-119::FRT*::galk::FRT* template for recombineering and biolistics

Fosmid based recombineering has become a popular method for generating fluorescent expression reporters that retain the cis-acting regulatory features of a gene of interest. To facilitate the identification of successfully targeted recombination events, Tursun et al. (2009) have developed a toolkit of recombineering cassettes carrying fluorescent gene sequences and the selectable galK marker flanked by FRT sites. To expand the versatility of such recombineered fosmids for use in biolistics (Praitis et al., 2001), we have generated the pPK719 plasmid, which carries a complementary cassette consisting of a genomic copy of the C. briggsae unc-119 gene and the selectable galK marker flanked by FRT* sites in the presumptive 3’ UTR of the Cbr-unc-119 gene (Fig. 1). The Cbr-unc-119 gene region was cloned for this purpose because it is more compact than the Cel-unc-119 gene, yet remains capable of rescuing the Cel-unc-119 gene (Maduro and Pilgrim, 1996). The inclusion of FRT* sites in the Cbr-unc-119::FRT*::galk::FRT* cassette minimizes the occurrence of unwanted recombination events when performing sequential rounds of recombineering using galK flanked by other FRT sequences.

Table 1: Primers for amplifying the pPK719 cassette; pCC1FOS homology arms are shown in brackets. Note that the pCC1Fos homology sequence in PK951 is also present in the pBSKSII(+) vector backbone.

The 3.6 kb Cbr-unc-119::FRT*::galk::FRT* cassette, which is PCR amplified from pPK719 using the PK951/PK952 primer pair (Table 1), can be targeted by recombineering to insert between nts 281:282 in the pCC1FOS fosmid vector backbone. In other words, this cassette can be readily inserted in all fosmids contained in the C. elegans library developed by Don Moerman and colleagues. We have tested the construct in fosmid recombineering and have shown that it rescues unc-119(ed3) mutants. The clone will be made available at http://www.Addgene.com.


Copy of unc-119 cassette-h
Figure 1: Cbr-unc-119::FRT*::galk::FRT* cassette contained in plasmid pPK719.


Maduro M, and Pilgrim D. (1996). Conservation of function and expression of unc-119 from two Caenorhabditis species despite divergence of non-coding DNA. Gene 183, 77-85. PubMed

Praitis V, Casey E, Collar D, and Austin J. (2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217-26. PubMed

Tursun B, Cochella L, Carrera I, and Hobert O. (2009). A toolkit and robust pipeline for generation of fosmid-based reporter genes in C. elegans. PLoS One 4, e4625. PubMed

Published: June 25, 2012 in