Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method
1 Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama, Japan
3 Saitama University Brain Science Institute, Saitama, Japan
BMC Biotechnology 2012, 12:1 doi:10.1186/1472-6750-12-1Published: 5 January 2012
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.
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.