High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii.
High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii.
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DOI：
10.3791/54831
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发表时间：
2016-12-05
期刊：
影响因子：
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通讯作者：
Cross FR
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文献类型：
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作者：
Breker M;Lieberman K;Tulin F;Cross FR
系统的鉴定和表征遗传扰动已被证明是有用的破译基因功能和细胞途径。然而，传统的永久基因删除方法不能应用于必需基因。我们率先收集了约70种温度敏感（ts）致死性突变体，用于研究单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtitii）的细胞周期调控。这些突变确定了必需基因，并且ts等位基因可以通过温度变化有条件地失活，为鉴定和分析必需功能提供了有价值的工具。如果突变集合接近全面，那么它们就更有价值，因为分散的集合可能会错过重要的组件。然而，这需要高效地收集大量突变体，特别是在宽目标筛选中。在这里，我们描述了一个机器人为基础的管道产生的致命突变体和分析其表型衣藻。这种技术可以应用于任何生长在琼脂上的微生物。我们已经收集了3000多个ts突变，可能包括大多数或所有细胞必需途径的突变，包括大约200个新的候选细胞周期突变。随后这些突变体的分子和细胞特性将为植物细胞生物学提供新的见解；一个全面的突变体收集是一个必要的先决条件，以确保覆盖广泛的生物学途径。这些方法与下游遗传学和生物信息学程序集成，以有效地绘制和鉴定超出本手稿范围的致病突变。
Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.