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

Kazuki Shirakawa,Guan Huang,Akira Ito,Masamichi Kamihira,Yoshinori Kawabe,M Shimada

doi:10.1051/matecconf/202133307003

Abstract

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 loxPs 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.

Highlights

Photosynthetic microalgae have attracted great interest as a sustainable green cell factory and have the potential to revolutionize many biotechnology fields including nutrition, pharmaceuticals and biofuels (Rasala and Mayfield, 2015)
Random integration of transgene into the nuclear genome of microalgae causes gene silencing of heterologous transgene expression depending on the insertion position
Integration efficiency of Cremediated reaction was dependent on the amount of Cre expression vector (Kameyama et al, 2010)

Summary

Introduction

Photosynthetic microalgae have attracted great interest as a sustainable green cell factory and have the potential to revolutionize many biotechnology fields including nutrition, pharmaceuticals and biofuels (Rasala and Mayfield, 2015). Engineered microalgae can be expected to be more useful in bioenergy and recombinant protein production. To generate genetically engineered microalgae, a foreign DNA fragment should be incorporated into the cell nuclear or chloroplast genome through successful transformation events. Protein expression from the nuclear genome has the advantage that extracellular secretion and appropriate post-translational modification can be performed, the expression level from the nuclear genome tends to be low compared to the chloroplast expression level. Random integration of transgene into the nuclear genome of microalgae causes gene silencing of heterologous transgene expression depending on the insertion position. Construction of a stable gene expression system is desired (Scranton et al, 2015)

Methods

Results

Conclusion