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

Eriko Kage-Nakadai, Hiroyuki Kobuna, Osamu Funatsu, Muneyoshi Otori, Keiko Gengyo-Ando, Sawako Yoshina, Sayaka Hori, Shohei Mitani

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

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.

Original languageEnglish
Article number1
JournalBMC Biotechnology
Volume12
DOIs
Publication statusPublished - 2012 Jan 5

ASJC Scopus subject areas

  • Biotechnology

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