Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Eukaryot Microbiol. 2011 Jan-Feb;58(1):37-42. doi: 10.1111/j.1550-7408.2010.00517.x. Epub 2010 Dec 3.
We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.
我们开发了一种使用荧光激活细胞分选仪 (FACS) 在化学定义培养基 (CDM) 中克隆纤毛虫 Tetrahymena thermophila 细胞的方法。虽然 T. thermophila 是一种模式单细胞真核生物，但在其克隆过程中仍然存在两个主要技术难题。首先，T. thermophila 在 CDM 中的低密度下无法增殖，特别是如果接种物包含单细胞。其次，一般的克隆方法耗时且通量低。在这里，我们通过添加牛血清白蛋白来修改 CDM，这有助于从密度为 10 细胞/ml（1 个细胞/100 μl）的接种物中生长。此外，我们应用了 FACS 来分离单细胞。我们表明，有可能根据吞噬荧光珠的存在与否来分离细胞群体，并通过 FACS 在改良的 CDM 中分离单个细胞。我们的技术允许直接分离单个细胞，并促进克隆株的建立。
