The development of methods to introduce exogenous DNA into animals has allowed for diverse genetic manipulations in many organisms. In Caenorhabditis elegans
, transgenic strains are typically generated by injecting DNA into the syncytial germ cells of the hermaphrodite gonad to form multi-copy extrachromosomal arrays [1
]. These transgenes are semi-stable; transgenic animals are mosaic in that some cells lose the extrachromosomal array, and transmission of arrays to the next generation is partial [2
]. Extrachromosomal arrays contain hundreds of copies of the injected DNA [1
], leading to silencing of the transgene expression in the germline [3
]. Although extrachromosomal arrays can be integrated into the chromosomes by gamma-ray irradiation or ultraviolet (UV) [4
], integrated arrays still contain a high copy-number of transgenes that seldom escape gene silencing.
Methods using microparticle bombardment were developed to create low-copy chromosomal integrated lines [6
]. The biolistic technique allows for direct integration of small amounts of exogenous DNA into the chromosomes, avoiding the formation of extrachromosomal arrays. Not every bombardment, however, produces integrant animals because of the low frequency of events, although large number of animals can be bombarded at once (~104
]. More recently, techniques using Mos1 transposons were developed and are frequently used to generate single-copy gene insertions [7
]. This technique, called Mos1-mediated single-copy insertion (MosSCI), is based on homologous recombination: A double-stranded break in the chromosome mediated by Mos1 excision is repaired with an exogenously supplied template carrying the gene of interest and homology arms, generating the designed single copy insertion [7
]. MosSCI methods, in which a recipient strain carrying a Mos1 element is microinjected with a targeting vector and a Mos1 transposase expression vector, exhibit a high frequency of insertion, and injection of 20 worms is enough to obtain integrant animals [7
]. In the MosSCI method, large sized targeting vectors that contain positive selection marker, 5'- and 3'- homology arms, and gene of interest must be constructed for each insertion, although a Gateway-compatible tool kit for MosSCI has been developed [8
In reverse genetic studies, ultraviolet trimethylpsoralen (UV/TMP), which induces a small deletion in the chromosomes, has been widely used to generate deletion mutants [9
]. In addition to deletions, insertions of unexpected DNA fragments are often observed, suggesting that non-homologous DNA is used as a template in end-joining repair mechanisms. UV irradiation (wavelength 365 nm) and TMP treatment has a higher mutation frequency and less rearrangement of chromosomes, such as inversion and translocation, compared to UV irradiation (wavelength 254 nm), which is used for insertion of extrachromosomal arrays [5
In the present study, we developed a technique using UV/TMP that produces single- or low- copy gene integrations from extrachromosomal arrays. In this method, we used a positive selection marker flanked by LoxP sites, allowing the marker to be excised by the Cre recombinase.