Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan.
Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe 651-2492, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan; Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan.
Gene. 2014 Jan 25;534(2):249-55. doi: 10.1016/j.gene.2013.10.049. Epub 2013 Nov 1.
A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2,000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.
一个嘌呤霉素 N-乙酰转移酶基因（pac）被广泛用作真核基因操作的选择标记。然而，尽管该生物体内可用于选择的标记有限，但它从未在纤毛虫嗜热四膜虫中用于分子研究。为了将 pac 用作嗜热四膜虫的标记基因，对 pac 基因的核苷酸序列进行了修饰，使其符合嗜热四膜虫中最优选的密码子使用。在嗜热四膜虫中表达的这种密码子优化的 pac 基因赋予转化细胞对 2000μg/ml 盐酸嘌呤霉素的抗性，而野生型细胞在 200μg/ml 时生长完全受到抑制。此外，用密码子优化的 pac 和 MTT1 启动子构建的表达盒有效地用于通过将盒融合到靶基因座中来标记感兴趣的内源蛋白的实验。这些结果表明，pac 可用于嗜热四膜虫的分子研究中作为选择标记。
