EAGER: Rapid evolution of enhancer DNA sequences in Drosophila

在动物生长和发育过程中，受精卵细胞必须分裂并产生不同类型的细胞（例如肌肉，皮肤，神经元）。这个过程中的错误会导致人类的发育缺陷和疾病。为了了解动物的发育程序是如何由其DNA编码的，引入DNA中的变化并分析其对发育的影响是有用的。然而，目前，在多细胞动物中一次只能产生和分析少数遗传变化。为了克服这一限制，北卡罗来纳州州立大学（NCSU）的研究人员将通过帮助控制苍蝇胚胎基因表达的小DNA片段的快速进化来快速探索细胞如何适应它们的命运，这是其他动物的一个很好的模型。同时，研究人员将开发一种用于高通量分析苍蝇胚胎发育的设备。该项目将由NCSU的研究生作为他们研究培训的一部分进行，其成果将通过研讨会和期刊文章进行交流。小的、有针对性的DNA片段的快速进化将通过在活果蝇细胞和胚胎中重复的转录、逆转录和重新整合来实现。逆转录的易错过程将突变引入DNA序列，该DNA序列被设计为驱动GFP标记的报告基因的表达。为了筛选大量果蝇胚胎中影响基因表达的突变，将构建由微流控阵列和自动成像方案组成的高通量平台。活胚胎将被成像，机器学习算法将检测和分类基因表达模式改变的胚胎。将收获分选的胚胎用于进一步研究。除了这项研究之外，高通量平台的开发将使果蝇胚胎和其他动物物种的筛选方案成为可能。该项目由分子和细胞生物科学部-遗传机制和系统以及合成生物学集群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

During animal growth and development, fertilized egg cells must divide and give rise to distinct types of cells (e.g. muscle, skin, neuron). Errors in this process result in developmental defects and disease in humans. In order to understand how an animal’s developmental program is encoded by its DNA, it is useful introduce changes in the DNA and analyze the effects on development. At present, however, it is only possible to generate and analyze a few genetic changes at a time in multicellular animals. To overcome this limitation, researchers at North Carolina State University (NCSU) will rapidly probe how cells adopt their fates by quick evolution of small DNA segments that help control gene expression in fly embryos, a good model for other animals. In parallel, the researchers will develop a device for high-throughput analysis of fly embryo development. This project will be undertaken by graduate students at NCSU as part of their research training, and the outcomes will be communicated through workshops and journal articles.The rapid evolution of small, targeted stretches of DNA will be achieved through repeated rounds of transcription, reverse transcription, and reintegration in live Drosophila cells and embryos. The error-prone process of reverse transcription will introduce mutations into a DNA sequence that is designed to drive the expression of a GFP-tagged reporter gene. To screen a large number of Drosophila embryos for mutations that affect gene expression, a high-throughput platform consisting of a microfluidic array and automated imaging protocols will be constructed. Live embryos will be imaged, and machine learning algorithms will detect and sort embryos that have altered gene expression patterns. The sorted embryos will be harvested for further study. Beyond this study, the development of the high-throughput platform will enable screening protocols in Drosophila embryos, and also in other animal species. This technology can open new avenues in biological design and engineering.This project is jointly funded by the Division of Molecular and Cellular Biosciences - Genetic Mechanisms and Systems and Synthetic Biology Clusters.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.