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首页> 外文期刊>Gene: An International Journal Focusing on Gene Cloning and Gene Structure and Function >Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe.
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Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe.

机译：在粟酒裂殖酵母中使用重组酶介导的盒交换进行基因标记和基因替换。

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摘要

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.
机译：Cre / lox位点特异性重组系统为基因操作提供了重要工具。在这里，我们介绍了使用Cre重组酶介导的盒式交换（RMCE）进行基因标记和基因置换的有效方法。该盒由粟酒裂殖酵母ura4（+）选择标记组成，其一侧为野生型loxP位点，另一侧为修饰的异源lox位点（loxM3）。盒式磁带很稳定，因为侧翼lox位点无法彼此重组。在将所述盒整合到所选的染色体基因座之后，通过引入Cre-表达质粒来实现交换，所述Cre-表达质粒包含含有所需标签或基因序列的等效盒。使用5-氟乳清酸试剂（5-FOA）通过尿嘧啶原养型选择重组子。盒交换系统提供了在相同基因座的重复整合，允许快速有效地整合不同的蛋白质标签或基因序列。我们已经建立了一系列试剂并通过用yEGFP和yEGFP衍生物yECFP和yECitrine端标记S. pombe rad4和swi1基因并通过转移这两个基因的编码序列来验证实用性。

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