HORI Sayaka
   Department   School of Medicine, School of Medicine
   Position   Assistant Professor
Article types Original article
Language English
Peer review Peer reviewed
Title Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method.
Journal Formal name:BMC biotechnology
Abbreviation:BMC Biotechnol
ISSN code:1472-6750(Electronic)1472-6750(Linking)
Volume, Issue, Page 12,1頁
Author and coauthor Kage-Nakadai Eriko, Kobuna Hiroyuki, Funatsu Osamu, Otori Muneyoshi, Gengyo-Ando Keiko, Yoshina Sawako, Hori Sayaka, Mitani Shohei
Publication date 2012/01
Summary BACKGROUND:Transgenic strains of CaBACKGROUND:enorhabditis 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.
DOI 10.1186/1472-6750-12-1
Document No. 22217006