Efficient and rapid exact gene replacement without selection.
Efficient and rapid exact gene replacement without selection.
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DOI：
10.1002/yea.1822
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发表时间：
2011-02
期刊：
影响因子：
2.6
通讯作者：
Pecani, Kresti
中科院分区：
文献类型：
--
作者：
Cross, Frederick R.;Pecani, Kresti
我们描述了一种在芽殖酵母中进行精确基因替换的高效方法。在整个细胞群体中诱导快速和有效的重组导致至少50%的重组体经历了内源拷贝到特定突变等位基因的特定转换，没有剩余的标记或外源DNA的残余，没有选择。为了实现这一点，在GAL-HO MATa-inc背景下，将替换等位基因的部分拷贝和HO切割位点安装在野生型位点附近。HO诱导导致近定量的位点切割和重组/基因转换，导致野生型的再生，或将内源等位基因转换为突变体，同时删除中间标记序列，产生精确的替换。消除了对稀有重组的选择（超过数天）的需要，消除了对第二位点抑制基因突变的担忧，并且还允许对致命基因替换进行直接表型分析，而不需要一种方法来使致死性有条件，或者使用与内源性启动子相比强度未知的调节启动子。为了验证这种方法，我们尝试了两种已知的致死性基因替换，用Cdk磷酸化位点突变的显性致死性基因替换非必需的CDH1基因，用两个隐性致死性基因替换必需的CDC28基因，其中一个含有早期停止密码子，另一个含有灭活CDC28激酶活性。我们还测试了未知表型后果的基因替换：用缺乏破坏盒的版本替换非必需的CLB3 b型细胞周期蛋白。
We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a specific switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type, or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations, and also allows direct phenotypic analysis even of lethal gene replacements without the need of a method to make the lethality conditional, or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites, and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another one inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box.
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影响因子：
11.4
作者：
HABER, JE
通讯作者：
HABER, JE
影响因子：
56.9
作者：
Visintin, R;Prinz, S;Amon, A
通讯作者：
Amon, A
影响因子：
5.3
作者：
SWEETSER, DB;HOUGH, H;NICKOLOFF, JA
通讯作者：
NICKOLOFF, JA
影响因子：
56.9
作者：
Zachariae, W;Schwab, M;Seufert, W
通讯作者：
Seufert, W
DOI：
10.1126/science.1191125
发表时间：
2010-07-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hicks WM;Kim M;Haber JE
通讯作者：
Haber JE