Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Biology, Plön, Germany.
PLoS Comput Biol. 2020 Nov 12;16(11):e1008354. doi: 10.1371/journal.pcbi.1008354. eCollection 2020 Nov.
Systematic knockout studies in mice have shown that a large fraction of the gene replacements show no lethal or other overt phenotypes. This has led to the development of more refined analysis schemes, including physiological, behavioral, developmental and cytological tests. However, transcriptomic analyses have not yet been systematically evaluated for non-lethal knockouts. We conducted a power analysis to determine the experimental conditions under which even small changes in transcript levels can be reliably traced. We have applied this to two gene disruption lines of genes for which no function was known so far. Dedicated phenotyping tests informed by the tissues and stages of highest expression of the two genes show small effects on the tested phenotypes. For the transcriptome analysis of these stages and tissues, we used a prior power analysis to determine the number of biological replicates and the sequencing depth. We find that under these conditions, the knockouts have a significant impact on the transcriptional networks, with thousands of genes showing small transcriptional changes. GO analysis suggests that A930004D18Rik is involved in developmental processes through contributing to protein complexes, and A830005F24Rik in extracellular matrix functions. Subsampling analysis of the data reveals that the increase in the number of biological replicates was more important that increasing the sequencing depth to arrive at these results. Hence, our proof-of-principle experiment suggests that transcriptomic analysis is indeed an option to study gene functions of genes with weak or no traceable phenotypic effects and it provides the boundary conditions under which this is possible.
系统敲除研究在小鼠中表明，很大一部分基因替换没有表现出致死或其他明显的表型。这导致了更精细的分析方案的发展，包括生理、行为、发育和细胞学测试。然而，对于非致死性敲除，转录组分析尚未得到系统评估。我们进行了功效分析，以确定即使在转录水平发生微小变化也能可靠追踪的实验条件。我们将其应用于两条基因敲除系，这些基因目前还没有功能。由两个基因表达最高的组织和阶段提供的专门表型测试表明，这些测试表型受到的影响很小。对于这些阶段和组织的转录组分析，我们使用了先前的功效分析来确定生物重复的数量和测序深度。我们发现，在这些条件下，敲除对转录网络有显著影响，数千个基因表现出微小的转录变化。GO 分析表明，A930004D18Rik 通过参与蛋白质复合物参与发育过程，而 A830005F24Rik 参与细胞外基质功能。对数据的抽样分析表明，增加生物重复的数量比增加测序深度更重要，才能得到这些结果。因此，我们的原理验证实验表明，转录组分析确实是研究具有微弱或无法追踪的表型效应的基因的基因功能的一种选择，它提供了这种方法可行的边界条件。
