Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System

The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct markerfree transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble( linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wildtype strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.