Faculty of Life Sciences, Manchester University, Manchester, UK.
Yeast. 2010 Sep;27(9):765-75. doi: 10.1002/yea.1774.
In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre-loxP system, so that they can be used with natural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE-hphNT1-loxRE and lox2272-natNT2-lox2272 gene-disruption cassettes can be present in the yeast genome together with the widely used loxP-marker gene-loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin-resistant Cre-expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon-limited resources.
在这项工作中，我们开发了可用于标准实验室酵母菌株（如 cre-loxP 系统）的分子工具，以便它们可以与天然和工业同型酵母物种一起使用。我们构建了新一代显性盒，带有突变的loxP 位点（loxLE 和 lox2272）和可选择的药物标记物，以创建异核菌株和营养缺陷型突变体，而不会产生染色体重排的风险。我们已经表明，我们新开发的loxLE-hphNT1-loxRE 和 lox2272-natNT2-lox2272 基因敲除盒可以与广泛使用的loxP-标记基因-loxP 盒一起存在于酵母基因组中，而lox 序列之间不会发生任何重组。此外，我们还开发了一种新的抗磷霉素 Cre 表达载体（用于同时切除多个标记物）和两个新的含有 Hygromycin B 和 CloNAT 作为选择标记物的标准 loxP 缺失盒。为了验证这些盒，我们创建了异核营养缺陷型 S. cerevisiae 菌株，而不会产生大规模染色体重排的风险，并且我们展示了一个来自具有不同适应碳限制资源的亲本的种内杂种适应性研究的例子。
