Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
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DOI：
10.1371/journal.pone.0163950
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Lindquist S
中科院分区：
文献类型：
--
作者：
Landgraf D;Huh D;Hallacli E;Lindquist S
用荧光蛋白进行基因标记通常用于研究蛋白质在其细胞环境中的定位和动态。理想情况下，在目的基因N-端或C-末端的内源性位点上插入一个荧光标记，而不会干扰包括5‘和3’非翻译区(UTR)在内的调控序列，也不会引入任何可能产生不可预测的转录或翻译效应的外来的不需要的“SCAR”序列。我们提出了一种可靠、低成本和高效的方法，用于在酵母中构建这种无疤痕的C端和N端与荧光蛋白的融合。该方法依靠顺序正反选择，利用两步法聚合酶链式反应组装的长侧翼区整合盒，提高同源重组频率。该方法还可以对必需基因进行无疤痕标记，而不需要补充质粒。为了进一步简化高通量菌株的构建，我们对引物进行了计算自动化设计，将引物设计代码应用于萌芽酵母酿酒酵母(S.cerevisiae)和裂殖酵母裂殖酵母(S.pombe)的所有开放阅读框(ORF)，并在此提供了计算序列。为了阐明酿酒酵母中无疤痕的N-端和C-端基因标记方法，我们用酵母密码子优化的mNeonGreen或mCherry标记了包括E3泛素连接酶RSP5、蛋白酶体亚单位PRE1和11个Rab GTP酶在内的各种基因；其中几个代表了必需基因。我们还利用kanMX6和HSV1tk以及ura4分别作为正选择标记和负选择标记，在远缘生物S.pombe中实现了无疤痕基因末端标记方法。本文提出的无疤痕基因标记方法广泛适用于可视化和研究活细胞中蛋白质的功能作用。
Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.7
作者：
Booher, Keith R.;Kaiser, Peter
通讯作者：
Kaiser, Peter
影响因子：
11.4
作者：
Abruzzi, Katharine C.;Belostotsky, Dmitry A.;Rosbash, Michael
通讯作者：
Rosbash, Michael
DOI：
10.1073/pnas.83.24.9373
发表时间：
1986-12-01
影响因子：
11.1
作者：
FREIER, SM;KIERZEK, R;TURNER, DH
通讯作者：
TURNER, DH