Dave Drashti, Wloga Dorota, Gaertig Jacek
Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA.
Methods Cell Biol. 2009;93:1-20. doi: 10.1016/S0091-679X(08)93001-6. Epub 2009 Dec 4.
Tetrahymena thermophila has emerged as an excellent protist model for studies on cilia that are based on reverse genetic approaches. In Tetrahymena, genes can be routinely disrupted by the DNA homologous recombination. We present established protocols for the manipulation of genes in either the germline micronucleus or the somatic macronucleus. A detailed protocol is provided for the construction of heterokaryon strains that carry a gene disruption only in the micronucleus. Heterokaryon strain can be propagated like wild-type cells, and ciliary phenotypes can be expressed on demand by mating. We describe methods that can be used for disruption of multiple genes. We include protocols for the generation and maintenance of Tetrahymena cells that either lack cilia or have paralyzed cilia.
嗜热四膜虫已成为基于反向遗传学方法研究纤毛的优秀原生生物模型。在嗜热四膜虫中，基因可通过DNA同源重组常规性地被破坏。我们展示了用于在生殖系小核或体细胞大核中操纵基因的既定方案。提供了一个详细方案，用于构建仅在小核中携带基因破坏的异核体菌株。异核体菌株可以像野生型细胞一样繁殖，并且通过交配可按需表达纤毛表型。我们描述了可用于破坏多个基因的方法。我们还包括了用于生成和维持无纤毛或纤毛麻痹的嗜热四膜虫细胞的方案。
