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Genetically modified microalgae have been expected to be a useful tool for bioenergy and recombinant protein production. However, random integration of transgene in the microalgae nuclear genome is susceptible to gene silencing of heterologous gene expression. Here, we attempted to perform targeted gene integration into a pre-determined nuclear genomic site of  Chlamydomonas reinhardtii  using Cre/ loxP  recombination system for stable transgene expression. We constructed an expression vector plasmid encoding reporter genes (zeocin resistant gene and green fluorescent protein gene;  Zeo-2A-GFP ) and mutated  loxP  to generate founder cells. A donor vector encoding  IFNα-4  and paromomycin resistant genes flanked by corresponding mutated  loxP s was constructed and introduced into founder cells together with a Cre expression vector. The optimal ratio of donor vector to Cre expression vector was determined by counting the number of paromomycin resistant colonies. For the established clones, the targeted integration was confirmed by genomic PCR using various specific primer sets. Target genes in the donor vector could be integrated into the expected genomic site of  C. reinhardtii  using Cre/ loxP  system. RT-PCR revealed that  IFNα-4  was expressed in five independent transgenic cell lines tested. This result suggests that Cre-based cell engineering is a promising approach to generate smart microalgae expressing foreign genes.

Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxP Recombination System