中国科学院机构知识库网格
Chinese Academy of Sciences Institutional Repositories Grid
Mazf as a counter-selectable marker for unmarked genetic modification of pichia pastoris

文献类型:期刊论文

作者Yang, Junjie1,2; Jiang, Weihong1; Yang, Sheng1,3
刊名Fems yeast research
出版日期2009-06-01
卷号9期号:4页码:600-609
关键词Pichia pastoris Mazf Counter-selectable marker Genetic modification
ISSN号1567-1356
DOI10.1111/j.1567-1364.2009.00503.x
通讯作者Yang, sheng(syang@sibs.ac.cn)
英文摘要In this study, we demonstrate a novel method for unmarked genetic modification of the methylotrophic yeast pichia pastoris, in which the escherichia coli toxin gene mazf was used as a counter-selectable marker. mazf was placed under the tightly controlled aox1 promoter, and the induced expression of mazf in p. pastoris halted cell growth. a modular plasmid was constructed in which mazf and a zeocin resistance gene acted as counter-selectable and active-selectable markers, respectively, and the mazf-zeor cassette was flanked by two direct repeats for marker recycling. linearized delivery vectors constructed from the modular plasmid were integrated into the p. pastoris genome via homologous recombination, introducing genetic modifications. upon counter-selection with methanol medium, which induces the aox1 promoter, the markers were recycled efficiently via homologous recombination between the direct repeats. we used this method successfully to knock-out the arg1 and met2 genes, knock-in a green fluorescent protein expression cassette, and perform site-directed mutagenesis on the arg1 gene, all without introducing unwanted selection markers. the novel method allows repeated use of the selectable marker gene for multiple modifications and will be a useful tool for p. pastoris studies.
WOS关键词MESSENGER-RNA INTERFERASE ; SINGLE PROTEIN-PRODUCTION ; PROGRAMMED CELL-DEATH ; ESCHERICHIA-COLI ; BUDDING YEAST ; DISRUPTION ; CLONING ; SYSTEM ; EXPRESSION ; SELECTION
WOS研究方向Biotechnology & Applied Microbiology ; Microbiology ; Mycology
WOS类目Biotechnology & Applied Microbiology ; Microbiology ; Mycology
语种英语
WOS记录号WOS:000265934800009
出版者WILEY-BLACKWELL PUBLISHING, INC
URI标识http://www.irgrid.ac.cn/handle/1471x/2397862
专题中国科学院大学
通讯作者Yang, Sheng
作者单位1.Chinese Acad Sci, Key Lab Synthet Biol, Inst Plant Physiol & Ecol, Shanghai Inst Biol Sci, Shanghai 200032, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing, Peoples R China
3.Chinese Acad Sci, Huzhou Res Ctr Ind Biotechnol, Shanghai Inst Biol Sci, Huzhou, Peoples R China
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GB/T 7714
Yang, Junjie,Jiang, Weihong,Yang, Sheng. Mazf as a counter-selectable marker for unmarked genetic modification of pichia pastoris[J]. Fems yeast research,2009,9(4):600-609.
APA Yang, Junjie,Jiang, Weihong,&Yang, Sheng.(2009).Mazf as a counter-selectable marker for unmarked genetic modification of pichia pastoris.Fems yeast research,9(4),600-609.
MLA Yang, Junjie,et al."Mazf as a counter-selectable marker for unmarked genetic modification of pichia pastoris".Fems yeast research 9.4(2009):600-609.

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来源:中国科学院大学

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