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Abstract：
Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking.
In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth.
The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user\'s requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.
In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth.
The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user\'s requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.
摘要：
基于自动图像的显微镜平台的进步与高通量液体工作流程相结合，促进了利用秀丽隐杆线虫等多细胞模型生物来识别遗传相互作用的大型屏幕的设计。治疗药物或疾病调节剂。然而,对必需基因的分析已经滞后，因为致命或无菌突变是高通量方法的瓶颈，并且缺乏一种系统的方法来分析多细胞生物中必需基因的遗传相互作用。
在秀丽隐杆线虫中,非条件致死突变可以用染色体平衡保持杂合性，通常在咽部表达绿色荧光蛋白（GFP）。然而,基因表达或功能通常通过使用标记有相同荧光团的荧光报道分子来监测，对感兴趣的蠕虫种群进行排序提出了挑战，特别是在幼体早期。这里,我们开发了一种分选策略，能够在第二个幼虫阶段从GFP平衡的动物中选择携带GFP应激报道分子的纯合突变体。因为排序不是完全没有错误的，我们开发了一个自动化的高通量图像分析协议，识别和丢弃携带染色体平衡器的动物。我们证明了在功能性基因组RNA干扰（RNAi）筛选中结合纯合致死突变体的分选和自动图像分析的实验有用性与线粒体阻抑素（PHB）遗传相互作用的基因。缺乏PHB会导致胚胎致死，而纯合PHB缺失突变体由于母体的贡献而发育成不育的成体，并强烈诱导线粒体未折叠蛋白反应（UPRmt）。在具有人类直向同源物的秀丽隐杆线虫基因的全染色体RNAi筛选中，我们发现了影响UPRmt和生长的已知和新的PHB遗传相互作用物。
本文提出的方法允许以高通量方式研究平衡的致死突变。它可以很容易地适应取决于用户的要求，应该作为一个有用的资源，为C.elegans社区探索新的生物学方面的基本线虫基因，以及产生更全面的遗传网络。
在秀丽隐杆线虫中,非条件致死突变可以用染色体平衡保持杂合性，通常在咽部表达绿色荧光蛋白（GFP）。然而,基因表达或功能通常通过使用标记有相同荧光团的荧光报道分子来监测，对感兴趣的蠕虫种群进行排序提出了挑战，特别是在幼体早期。这里,我们开发了一种分选策略，能够在第二个幼虫阶段从GFP平衡的动物中选择携带GFP应激报道分子的纯合突变体。因为排序不是完全没有错误的，我们开发了一个自动化的高通量图像分析协议，识别和丢弃携带染色体平衡器的动物。我们证明了在功能性基因组RNA干扰（RNAi）筛选中结合纯合致死突变体的分选和自动图像分析的实验有用性与线粒体阻抑素（PHB）遗传相互作用的基因。缺乏PHB会导致胚胎致死，而纯合PHB缺失突变体由于母体的贡献而发育成不育的成体，并强烈诱导线粒体未折叠蛋白反应（UPRmt）。在具有人类直向同源物的秀丽隐杆线虫基因的全染色体RNAi筛选中，我们发现了影响UPRmt和生长的已知和新的PHB遗传相互作用物。
本文提出的方法允许以高通量方式研究平衡的致死突变。它可以很容易地适应取决于用户的要求，应该作为一个有用的资源，为C.elegans社区探索新的生物学方面的基本线虫基因，以及产生更全面的遗传网络。
