Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method

We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans.Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.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) [4,5], 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