基因修饰人多能干细胞的高效单克隆建系方法

目标:提供一种能够显著提高慢病毒稳定转染人多能干细胞的方法,并建立一种简便无损的转染细胞筛选方法.方法:在慢病毒转染人多能干细胞过程,分别比较添加与不添加Y-27632情况下细胞形态的动态变化规律,以及细胞不同形态下对慢病毒颗粒的摄入能力差异,优化建立高效的慢病毒转染方法.随后,设计并研制可视化的简便显微操作装置,探索...

An Efficient Monoclonal Establishment Method of Genetically Modified Human Pluripotent Stem Cells

Objective: To provide a method that can significantly improve the stable transfection of lentivirus into human pluripotent stem cells, and establish a simple and a non-invasive screening method for transfected cells. Methods: In the process of lentivirus transfection of human pluripotent stem cells, we compared the dynamic changes of cell morphology with and without Y-27632, and the differences of lentivirus particles uptake ability under different cell morphologies, so as to optimize and establish an efficient lentivirus transfection method. After that, a visualized and simple micromanipulation device was designed and developed to explore the technology of picking up the transfected positive monoclone cells to establish a line with the aid of a fluorescence microscope, and establish a relatively simple new method for purification of transfected cells. Results: The morphology of normal cultured human pluripotent stem cells colony changed significantly 6 hours after Y-27632 was added. The cells were in loose colony showing a long spindle shape, and an increased cell surface area; The colonies returned to normal 6 hours after removal; In conventionally cultured pluripotent stem cells, lentivirus tended to enter the colony periphery or partial cells; after Y-27632 was treated for 6 hours in advance, the pluripotent stem cells showed a loose colony and a significant increased surface, making the lentivirus infect more evenly into the periphery and internal of the colony. It significantly improved the efficiency of lentiviral transfection. Using the capillary glass tube, we designed and manufactured independently a single colony selection device that was visualized under a microscope. With the aid of a microscope, the selection and establishment of successfully transfected colony can be easily performed in the laboratory. It can replace puromycin screening with certain cell damage and the flow cytometry which requires professional equipment. Conclusions: In the process of lentiviral transfection, the hESC / IPSC colonies cultured in conventional condition were relatively dense and resistant to lentivirus. Y-27632, a small molecule compound, made the hESC / IPSC colonies relatively loose in structure and increased the surface area, which significantly improved the susceptibility of cells to lentivirus and improved the infection efficiency; a simple and non-toxic micromanipulation device was successfully designed under conventional laboratory conditions, and it can effectively replace flow cytometry and drug screening, and realize the selection of cell clones and finally the establishment of cell lines.