Genome editing and high-throughput cell analysis platform

我们的提案需要我们迫切需要的设备套件，以跟上我们对基因组编辑和高内容成像工作流程的需求。IncuCyte SX1高通量成像平台是一种功能强大且经济实惠的解决方案，允许我们使用相位对比(无标记)成像和荧光标记探针执行各种活细胞分析。IncuCyte SX1在细胞培养孵化器中工作，因此确保几乎不受干扰地对细胞系进行长期成像。为了生产待用IncuCyte SX1检测的敲除、敲除和敲入细胞系，我们需要核转录因子4D。该仪器使用电脉冲将DNA、RNA和蛋白质直接输送到细胞质和细胞核中，同时保持高细胞活力。这项技术是提供纯化的Cas9核酸酶与sgRNA双链用于CRISPR基因组编辑的基准。对于大多数细胞系来说，成功的基因组编辑需要高达99%的转染率和高存活率。在核融合的下游，将使用IncuCyte SX1监测通过稀释克隆选择的单细胞克隆。最后，我们要求CellDrop消耗品无细胞计数器作为一种实用和经济的解决方案，以跟上我们HQP处理的不断增加的细胞培养量。*所需的设备将使我们的实验室能够在竞争激烈的国际研究环境中继续取得成功，并产生高影响力的工作。*这些设备将由我们的总部人员使用，以确保获得关于高度先进技术的实践培训机会。独特的设备和经过深思熟虑的技术知识将使我们的HQP在他们的项目和未来的职业选择中具有很强的竞争力。*这些设备将使我们实验室和部门其他实验室以及蒙特利尔研究社区的HQP受益。**

Our proposal requests the equipment suite we urgently need to keep up with our demand for genome editing and high-content imaging workflows. The IncuCyte SX1 high-throughput imaging platform is a powerful yet economical solution that allows us to perform a wide variety of live cell assays using phase contrast (label free) imaging and fluorescently labeled probes. The IncuCyte SX1 works inside a cell culture incubator and therefore guarantees virtually disturbance free, long-term imaging of cell lines. To produce the knock-out, knock-down, and knock-in cell lines to be assayed by the IncuCyte SX1, we request the Nucleofector 4D. The instrument uses electrical pulses to deliver DNA, RNA, and protein directly into the cytosol and nucleus while maintaining high cell viability. The technology is the benchmark in delivering purified Cas9 nuclease duplexed with sgRNA for CRISPR genome editing. Up to 99% transfection efficiency combined with high viability for most cell lines is required for successful genome editing. Downstream of nucleofection a selection of single cell clones by dilution cloning will be monitored using the IncuCyte SX1. Lastly, we are requesting a CellDrop consumable free cell counter as a practical and economical solution to keep up with the increased cell culture volume handled by our HQPs.******The equipment requested will enable our labs to continue to succeed in a highly competitive international research environment and produce high impact work. ******The equipment will be used by our HQPs, ensuring hands-on training opportunities on highly advanced technologies. The unique equipment and thought-after technology knowledge will enable our HQPs to be very competitive in their projects as well as their future career choices.***The equipment will benefit HQPs from our labs and from other labs in the department as well as the Montreal research community.**