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Molecules 2015, 20(6), 10110-10121; doi:10.3390/molecules200610110

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China
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Author to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Received: 10 April 2015 / Revised: 8 May 2015 / Accepted: 21 May 2015 / Published: 1 June 2015
(This article belongs to the Section Molecular Diversity)
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Abstract

The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cmr), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Bler) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes. View Full-Text
Keywords: antibiotic resistance marker gene; Aurantiochytrium limacinum; Cre/loxP site-specific recombination system; homologous recombination antibiotic resistance marker gene; Aurantiochytrium limacinum; Cre/loxP site-specific recombination system; homologous recombination
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Sun, H.; Chen, H.; Zang, X.; Hou, P.; Zhou, B.; Liu, Y.; Wu, F.; Cao, X.; Zhang, X. Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum. Molecules 2015, 20, 10110-10121.

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