Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum
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 in...
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doaj-53561070889545dda60cdae7188e3b9f2020-11-24T23:59:43ZengMDPI AGMolecules1420-30492015-06-01206101101012110.3390/molecules200610110molecules200610110Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinumHengyi Sun0Hao Chen1Xiaonan Zang2Pan Hou3Bingbing Zhou4Yuantao Liu5Fei Wu6Xiaofei Cao7Xuecheng Zhang8Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaKey Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, ChinaThe 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.http://www.mdpi.com/1420-3049/20/6/10110antibiotic resistance marker geneAurantiochytrium limacinumCre/loxP site-specific recombination systemhomologous recombination |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hengyi Sun Hao Chen Xiaonan Zang Pan Hou Bingbing Zhou Yuantao Liu Fei Wu Xiaofei Cao Xuecheng Zhang |
spellingShingle |
Hengyi Sun Hao Chen Xiaonan Zang Pan Hou Bingbing Zhou Yuantao Liu Fei Wu Xiaofei Cao Xuecheng Zhang Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum Molecules antibiotic resistance marker gene Aurantiochytrium limacinum Cre/loxP site-specific recombination system homologous recombination |
author_facet |
Hengyi Sun Hao Chen Xiaonan Zang Pan Hou Bingbing Zhou Yuantao Liu Fei Wu Xiaofei Cao Xuecheng Zhang |
author_sort |
Hengyi Sun |
title |
Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum |
title_short |
Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum |
title_full |
Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum |
title_fullStr |
Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum |
title_full_unstemmed |
Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum |
title_sort |
application of the cre/loxp site-specific recombination system for gene transformation in aurantiochytrium limacinum |
publisher |
MDPI AG |
series |
Molecules |
issn |
1420-3049 |
publishDate |
2015-06-01 |
description |
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. |
topic |
antibiotic resistance marker gene Aurantiochytrium limacinum Cre/loxP site-specific recombination system homologous recombination |
url |
http://www.mdpi.com/1420-3049/20/6/10110 |
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