Bergkessel Megan, Guthrie Christine, Abelson John
Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.
Methods Enzymol. 2013;533:43-55. doi: 10.1016/B978-0-12-420067-8.00005-2.
It is often useful to replace a small region of the yeast genome containing a gene of interest with a selectable marker. The selectable marker allows for easy identification of yeast cells that have successfully carried out the gene replacement, and functional consequences of the loss of that gene can then be assessed. The same technique can also be used for removing noncoding portions of the genome that may be of interest, such as promoters or 3' or 5' UTRs, and for introducing tags on the N- or C-termini of proteins (alternatively, see a marker-free yeast gene replacement method on Gene Knockouts, in vivo site-directed mutagenesis and Other Modifications Using the Delitto Perfetto System in Saccharomyces cerevisiae).
用一个可选择标记替换酵母基因组中包含感兴趣基因的小区域通常是很有用的。该可选择标记便于识别已成功进行基因替换的酵母细胞,进而可以评估该基因缺失的功能后果。同样的技术也可用于去除基因组中可能感兴趣的非编码部分,如启动子或3'或5'非翻译区,以及用于在蛋白质的N端或C端引入标签(另外,可参见酿酒酵母中使用完美诱变系统进行基因敲除、体内定点诱变和其他修饰的无标记酵母基因替换方法)。