A Fast and Reliable Method to Generate Pure, Single Cell-derived Clones of Mammalian Cells
Overview
Affiliations
Stable cell cloning is an essential aspect of biological research. All advanced genome editing tools rely heavily on stable, pure, single cell-derived clones of genetically engineered cells. For years, researchers have depended on single-cell dilutions seeded in 96- or 192-well plates, followed by microscopic exclusion of the wells seeded with more than or without a cell. This method is not just laborious, time-consuming, and uneconomical but also liable to unintentional error in identifying the wells seeded with a single cell. All these disadvantages may increase the time needed to generate a stable clone. Here, we report an easy-to-follow and straightforward method to conveniently create pure, stable clones in less than half the time traditionally required. Our approach utilizes cloning cylinders with non-toxic tissue-tek gel, commonly used for immobilizing tissues for sectioning, followed by trypsinization and screening of the genome-edited clones. Our approach uses minimal cell handling steps, thus decreasing the time invested in generating the pure clones effortlessly and economically. Graphical abstract: A schematic comparison showing the traditional dilution cloning and the method described here. Here, a well-separated colony (in the green box) must be preferred over the colonies not well separated (in the red box).
Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.
Zhou C, Wu Y, Ishidate F, Fujiwara T, Kengaku M J Cell Biol. 2024; 223(11).
PMID: 39115447 PMC: 11310688. DOI: 10.1083/jcb.202405032.
The importance of nuclear RAGE-Mcm2 axis in diabetes or cancer-associated replication stress.
Han Z, Andrs M, Madhavan B, Kaymak S, Sulaj A, Kender Z Nucleic Acids Res. 2023; 51(5):2298-2318.
PMID: 36807739 PMC: 10018352. DOI: 10.1093/nar/gkad085.