PhiC31 integrase for recombination-mediated single-copy insertion and genome manipulation in Caenorhabditis elegans. Issue 2 (15th November 2021)

• Record Type: Journal Article
• Authors: Yang, Fang-Jung; Chen, Chiao-Nung; Chang, Tiffany; Cheng, Ting-Wei; Chang, Ni-Chen; Kao, Chia-Yi; Lee, Chih-Chi; Huang, Yu-Ching; Hsu, Jung-Chen; Li, Jengyi; Lu, Meiyeh J; Chan, Shih-Peng; Wang, John
• Editors: Goldstein, B
• Is Part Of: Genetics. Volume 220:Issue 2(2022)
• Publication Date: 2021-11-15
• Subjects: phiC31 integrase -- recombination-mediated cassette exchange -- integration -- dual-fluorescent operon reporter -- inversion; Genetics -- Periodicals; 576.5
• DOI: 10.1093/genetics/iyab206
• Languages: English
• ISSNs: 0016-6731

Abstract

Abstract: Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans . We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(−) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.