Abstract
Although microalgae have valuable resources with great necessity in many biotechnological applications, no tools have been developed yet for a stable genetic transformation without antibiotic marker genes in these organisms. Chlorella is one of the most useful genus for biotechnological applications. The transfer of foreign DNA (vector or linear DNA cassette) into Chlorella by electroporation has very low stability and it is hard to screen the transformants without antibiotic marker genes. However, the marker genes have some disadvantages to host cells. To avoid the negative effects caused by the marker genes, we tried to develop a stable marker-free nuclear transformation system in Chlorella. For this, linear gene expression cassettes (LGEC) were constructed with functional domains, which are responsible for transformation, of SV40 large T antigen. The LGECs were transferred into Chlorella via electroporation and durability of the LGECs were confirmed in transgenic Chlorella. Transcription levels of the LGECs were also determined at different cell cycle sates. The LGECs integrated into the chromosomal DNA of Chlorella were stably replicated and were expressed successfully at G0-, G1-, and G2-phases. This study presents a stable marker-free nuclear transformation system with potential for biotechnological applications. Key words: Chlorella vulgaris, marker-free nuclear transformation, SV40 large T antigen, microalga.
Highlights
In recent years, microalgae have become valuable resources with its great necessity in many of biotechnological applications (Chu, 2012)
For the phosphorylation of the linear gene expression cassettes (LGECs)-A and -C, the primer pairs 35Sf/35Sr and 18Sf/18Sr were phosphorylated at 5’ end, and PCR was performed with these primer pairs using the LGEC-A or -C as a template
The DNA bands with exact size (843 bp) were amplified from eight colonies of transgenic C. vulgaris cells with LGEC-A, -B, -C, or -D and these results demonstrated that the LGEC were successfully transformed into C. vulgaris cells
Summary
Microalgae have become valuable resources with its great necessity in many of biotechnological applications (Chu, 2012). To improve microalgae for industrial application, various.
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