TY - JOUR
T1 - Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system
AU - Huang, Guan
AU - Kawabe, Yoshinori
AU - Shirakawa, Kazuki
AU - Akiyama, Tatsuki
AU - Kamihira, Masamichi
N1 - Funding Information:
We thank Edanz for editing the English text of a draft of this manuscript. This work was supported by a Grant-in-Aid for Scientific Research ( 20K05233 ) from the Japan Society for the Promotion of Science and a grant from the Nakamura Jishiro Ikueikai Foundation (Japan) .
Publisher Copyright:
© 2021 The Society for Biotechnology, Japan
PY - 2021/11
Y1 - 2021/11
N2 - The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.
AB - The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.
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U2 - 10.1016/j.jbiosc.2021.07.006
DO - 10.1016/j.jbiosc.2021.07.006
M3 - Article
C2 - 34420898
AN - SCOPUS:85113587244
VL - 132
SP - 469
EP - 478
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
SN - 1389-1723
IS - 5
ER -