Genome editing and high-throughput cell analysis platform

我们的提案要求提供我们迫切需要的设备套件，以满足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但经济的解决方案，使我们能够使用相差（无标记）成像和荧光标记探针进行各种活细胞测定。IncuCyte SX 1在细胞培养箱中工作，因此可以保证对细胞系进行无干扰的长期成像。为了产生待通过IncuCyte SX 1测定的敲除、敲低和敲入细胞系，我们要求使用Nucleofector 4D。该仪器使用电脉冲将DNA、RNA和蛋白质直接递送到细胞质和细胞核中，同时保持高细胞活力。该技术是递送纯化的Cas9核酸酶与sgRNA结合用于CRISPR基因组编辑的基准。 成功的基因组编辑需要高达99%的转染效率以及大多数细胞系的高活力。在核转染下游，将使用IncuCyte SX 1监测通过稀释克隆选择的单细胞克隆。最后，我们需要CellDrop免消耗细胞计数器作为一种实用且经济的解决方案，以跟上我们的HQP处理的细胞培养体积的增加。**所要求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HQP使用，以确保有机会获得关于高度先进技术的实践培训。独特的设备和经过深思熟虑的技术知识将使我们的HQP在他们的项目以及未来的职业选择中具有很强的竞争力。*该设备将有利于HQP从我们的实验室和其他实验室在部门以及蒙特利尔研究界。**

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.**