Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm
Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm
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DOI：
10.1039/c2ib20078c
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发表时间：
2013-01-01
影响因子：
2.5
通讯作者：
Lu, Hang
中科院分区：
文献类型：
--
作者：
Lee, Hyewon;Crane, Matthew M.;Lu, Hang
通过诱变进行的正向遗传筛选是鉴定模式生物如土栖自由生活线虫秀丽隐杆线虫（Caushabditis elegans）中靶途径中的调节因子的有力方法。elegans）。目前，手动显微镜是进行这种筛选的标准技术;然而，它是劳动密集型和耗时的，因为筛选需要对数千只动物进行成像。最近已经开发了微流体芯片以增加一些这样的实验的吞吐量;然而，这些芯片中的大多数是多层装置并且制造复杂，因此在制造和操作期间容易发生故障。此外，大多数分类决策都是手动做出的，并且用于分类的标准是主观的。为了克服这些局限性，我们开发了一种简单的单层微流控装置和一种自适应算法来做出分选决策。单层设备大大提高了可靠性，而使用自适应算法的定量分析允许识别产生表达细微变化的突变，这将很难用眼睛检测到。筛选标准是根据诱变种群而不是在实际筛选实验之前测量的单独对照种群设定的，以解释诱变蠕虫中基因表达的随机性和每日变化。此外，在每次实验中，阈值不断更新，以反映最大化的分类率和最小化的假阳性率之间的平衡。使用这个系统，我们筛选的突变体，改变了色氨酸羟化酶的表达水平，一个关键酶的5-羟色胺合成在CaMKII获得的功能背景。我们在筛选中发现了几个可能的突变体。此外，这种微流控系统和定量分析可以很容易地适用于研究其他途径在C。优美的
Forward genetic screening via mutagenesis is a powerful method for identifying regulatory factors in target pathways in model organisms such as the soil-dwelling free-living nematode Caenorhabditis elegans (C. elegans). Currently manual microscopy is the standard technique for conducting such screens; however, it is labor-intensive and time-consuming because screening requires imaging thousands of animals. Recently microfluidic chips have been developed to increase the throughput of some of such experiments; nonetheless, most of these chips are multilayer devices and complicated to fabricate and therefore prone to failure during fabrication and operation. In addition, most sorting decisions are made manually and the criteria used for sorting are subjective. To overcome these limitations, we developed a simple single-layer microfluidic device and an adaptive algorithm to make sorting decisions. The one-layer device greatly improves the reliability, while quantitative analysis with the adaptive algorithm allows for the identification of mutations that generate subtle changes in expression, which would have been hard to detect by eye. The screening criterion is set based on the mutagenized population, not separate control populations measured prior to actual screening experiments, to account for stochasticity and day-to-day variations of gene expression in mutagenized worms. Moreover, during each experiment, the threshold is constantly updated to reflect the balance between maximizing sorting rate and minimizing false-positive rate. Using this system, we screened for mutants that have altered expression levels of tryptophan hydroxylase, a key enzyme for serotonin synthesis in a CaMKII gain-of-function background. We found several putative mutants in this screen. Furthermore, this microfluidic system and quantitative analysis can be easily adapted to study other pathways in C. elegans.