Efficient gene targeting and removal of foreign DNA by homologous recombination in the picoeukaryote Ostreococcus

Abstract

These authors contributed equally. SUMMARYWith fewer than 8000 genes and a minimalist cellular organization, the green picoalga Ostreococcus tauri is one of the simplest photosynthetic eukaryotes. Ostreococcus tauri contains many plant-specific genes but exhibits a very low gene redundancy. The haploid genome is extremely dense with few repeated sequences and rare transposons. Thanks to the implementation of genetic transformation and vectors for inducible overexpression/knockdown this picoeukaryotic alga has emerged in recent years as a model organism for functional genomics analyses and systems biology. Here we report the development of an efficient gene targeting technique which we use to knock out the nitrate reductase and ferritin genes and to knock in a luciferase reporter in frame to the ferritin native protein. Furthermore, we show that the frequency of insertion by homologous recombination is greatly enhanced when the transgene is designed to replace an existing genomic insertion. We propose that a natural mechanism based on homologous recombination may operate to remove inserted DNA sequences from the genome.

Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton

“…2C); FTN-luc (WT-FTN), described above, and FTN-luc (ΔFtn), a line in which the luciferase is fused in frame to ferritin (24 kDa) at the native locus giving rise to a 90-kDa protein (30). A radiolabeled band at the expected size for the ferritin complex (480 kDa) was observed in WT but not in ΔFtn cells grown in the presence of 1 μM 55 Fe(III) citrate.…”

“…The iron-binding capacity of O. tauri ferritin was investigated using short-term (1-to 3-h) incubations of WT cells and a ΔFtn ferritin knock-out line (30) in the presence of 1 μM 55 Fe(III) citrate in Mf medium. We evidenced iron-binding proteins on blue native gels, using the mild detergent digitonin to avoid iron release from proteins (31).…”

“…The construction of the ferritin knock-out (ΔFtn), FTN-Luc knock-in translational reporters (WT and KO background), and NR knock-out (ΔNR) cells has been described elsewhere (30). Cells were grown at 20°C under a 12-h/12-h light/dark regime or under constant light for monitoring circadian expression unless otherwise stated.…”

“…In this study, we used O. tauri, a species which has emerged as a model organism thanks to the ease of culture and techniques for functional analyses such as genetic transformation and gene targeting by homologous recombination (28)(29)(30). We investigated the diel regulation of ferritin and iron-binding proteins in the field and under laboratory conditions.…”

Photoperiodism: The Calendar of Plants

“…The coccoid cells are haplonts with a rather simple cell structure: Just one chloroplast, one mitochondrion, one Golgi body besides the nucleus, no cell wall. The tiny genome (13 Mb, fewer than 8000 genes with a very low redundancy, see Lozano et al 2014) has been sequenced and biological information attached to the sequences (see http://www.geneontology.org/ GO.evidence.shtml and Corellou et al 2009). Under LD conditions, almost all genes were rhythmic (Monnier et al 2010).…”