Kumar Anuj
Department of Molecular, Cellular, and Developmental Biology, Life Sciences Institute, The University of Michigan, Ann Arbor, MI, USA.
Methods Mol Biol. 2008;416:117-29. doi: 10.1007/978-1-59745-321-9_8.
Transposons have long been recognized as useful laboratory tools facilitating genome-scale studies of gene function. Relative to traditional methods, transposon mutagenesis offers a rapid and economical means of generating large numbers of independent insertions in target DNA through minimal experimental manipulation. In particular, the transposon insertion library described here is an excellent tool for the analysis of gene function on a large scale in the budding yeast Saccharomyces cerevisiae. The transposon utilized in this library is multifunctional, such that the library can be used to screen for disruption phenotypes while also providing a means to generate epitope-tagged alleles and, in many cases, conditional alleles. Provided here are complete protocols by which the transposon insertion library may be used to screen for mutant phenotypes in yeast as well as accompanying protocols describing a means of identifying transposon insertion sites within strains of interest. In total, this insertion library is a singularly useful tool for genome-wide functional analysis, and the general approach is applicable to other organisms in which transforming DNA tends to integrate by homologous recombination.
转座子长期以来一直被认为是有助于进行基因功能全基因组规模研究的有用实验室工具。相对于传统方法，转座子诱变提供了一种快速且经济的手段，通过最少的实验操作在目标DNA中产生大量独立插入。特别是，这里描述的转座子插入文库是用于大规模分析酿酒酵母基因功能的优秀工具。该文库中使用的转座子具有多种功能，因此该文库可用于筛选破坏表型，同时还提供了一种产生表位标签等位基因的方法，并且在许多情况下还能产生条件等位基因。这里提供了完整的方案，通过这些方案可以使用转座子插入文库在酵母中筛选突变表型，以及伴随的方案，描述了一种在感兴趣的菌株中鉴定转座子插入位点的方法。总的来说，这个插入文库是全基因组功能分析的一个非常有用的工具，并且这种一般方法适用于其他通过同源重组使转化DNA倾向于整合的生物体。
