Image-guided robot for high-throughput microinjection of Drosophila embryos

项目摘要 该提案是为了响应NIH动物模型和相关生物学的发展而提交的。 研究材料（R21）计划。该提案开发了一种图像引导的机器人平台， 分子遗传工具和非遗传编码试剂的自动化递送 库、监测细胞过程的荧光染料、功能化磁珠或纳米颗粒， 数千个果蝇胚胎的实验。拟议的工作建立在最近 我们的合作小组开发了图像引导机器人系统， 与完整组织中的单细胞精确地连接。这两个具体目标提供了一个系统的 发展所提出的技术。AIM 1首先设计了一个机器人平台（“自动注射器”）， 在产卵板阵列中扫描并成像果蝇胚胎。我们将利用机器学习算法 用于胚胎的自动检测，随后进行阈值和形态分析以检测胚胎 质心和注释注射部位。在AIM 2中，我们将利用微处理器控制的流体回路， 将毫微微升至毫微微升体积的试剂程序性递送到单个胚胎中。我们将量化 通过比较转座子的存活率、生育力和转化率， PhiC31介导的转基因到手动显微注射数据集。最后，我们将展示有效的 SgRNA的递送和在Cas9存在下的诱变。该项目非常符合 通过设计一种新的工具来生产和改进动物模型。自动注射装置将 加速果蝇研究，使科学家能够进行新的实验和基因组规模 功能基因组学筛选目前效率太低或劳动密集型，无法在大型 规模，并可以另外实现其他新颖的未来应用。

PROJECT SUMMARY This proposal is submitted in response to the NIH Development of Animal Models and Related Biological Materials for Research (R21) program. The proposal develops an image-guided robotic platform that performs the automated delivery of molecular genetic tools and non-genetically encoded reagents such as chemical libraries, fluorescent dyes to monitor cellular processes, functionalized magnetic beads, or nanoparticles into thousands of Drosophila embryos in a single experimental session. The proposed work builds on recent engineering innovations in our collaborative group which has developed image-guided robotic systems that can precisely interface with single cells in intact tissue. The two Specific Aims provide for a systematic development of the proposed technologies. AIM 1 first engineers a robotic platform (‘Autoinjector’) that can scan and image Drosophila embryos in arrays of egg laying plates. We will utilize machine learning algorithms for automated detection of embryos, followed by thresholding and morphology analysis to detect embryo centroids and annotate injection sites. In AIM 2, we will utilize microprocessor-controlled fluidic circuits for programmatic delivery of femtoliter to nanoliter volumes of reagents into individual embryos. We will quantify the efficacy of the Autoinjector by comparing the survival, fertility, and transformation rates of transposon or PhiC31-mediated transgenesis to manual microinjection datasets. Finally, we will demonstrate the efficient delivery of sgRNAs and mutagenesis in the presence of Cas9. This project fits very well within the goals of the program by engineering a novel tool for producing and improving animal models. The Autoinjector will accelerate Drosophila research and empower scientists to perform novel experiments and genome-scale functional genomics screens that are currently too inefficient or labor intensive to be conducted on a large scale and may additionally enable other novel future applications.