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

Abstract

Background: Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single-or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. Results: 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. Conclusion: Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.

Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells

“…Transgenic strains with extra-chromosomal arrays were followed by expression of odr-1::gfp or odr-1::rfp (L'Etoile and Bargmann, 2000), which were injected at 100 ng/µl. ycIs11 was integrated using UV/TMP treatment (Kage-Nakadai et al, 2012). RNAi knockdown experiments were performed by feeding L4 animals bacteria expressing dsRNA from the Ahringer library (Source Bioscience) (Kamath et al, 2003) at 25°C.…”

Metal-induced neurodegeneration in C. elegans

“…Researchers have also found that this “gene gun” technique not only reproducibly allows for invariable expression levels and patterns that are difficult to obtain with extrachromosomal arrays, but that low-copy transgene expression does not get silenced in the germline (Praitis et al, 2001). Newer techniques to allow single- or low-copy transgene expression without bombardment are in the process of development, including the use of ultraviolent trimethylpsoralen (Kage-Nakadai et al, 2012). …”

Diversity of Fusarium species and mycotoxins contaminating pineapple

“…The fumonisin biosynthetic pathway has been well recognized and described (Proctor et al 2006) and FUM genes (particularly FUM1 ) have often been used for studies of fumonisin-producing fungi (Khaldi and Wolfe 2011; Proctor et al 2008; Stępień et al 2011a; Waalwijk et al 2004). Recently, the structure of the BEA biosynthetic gene cluster in F. proliferatum has been revealed (Zhang et al 2012). Moreover, two clusters responsible for the synthesis of fusarins and fusaric acid by F. verticillioides have been elucidated (Brown et al 2012).…”