Chilcoat N D, Elde N C, Turkewitz A P
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8709-13. doi: 10.1073/pnas.151243498. Epub 2001 Jul 3.
We report a pioneering approach using Tetrahymena thermophila that permits rapid identification of genes based on their null or hypomorphic phenotypes. This technique involves cell transformation with a library of plasmids that encode 26S ribosomal subunits containing short insertions. The insertions correspond to antisense sequences for a large number of genes. The majority of cells each acquires a single antisense sequence, which silences a single genomic locus. Because the insertion site within the ribosomal sequence is known, the silenced gene is easily amplified. We demonstrate that this approach can be used to identify genes required for dense core granule exocytosis.
我们报道了一种利用嗜热四膜虫的开创性方法，该方法可基于基因的无效或亚效表型快速鉴定基因。这项技术涉及用一个质粒文库对细胞进行转化，这些质粒编码含有短插入片段的26S核糖体亚基。这些插入片段对应于大量基因的反义序列。大多数细胞各自获得一个反义序列，该序列会使单个基因组位点沉默。由于核糖体序列内的插入位点是已知的，因此被沉默的基因很容易被扩增。我们证明这种方法可用于鉴定致密核心颗粒胞吐所需的基因。
