An approach for accelerated isolation of genetically manipulated cell clones with reduced clonal variability

• Published in: Journal of cell science Vol. 132; no. 6
• Main Authors: Casden, Natania, Behar, Oded
• Format: Journal Article
• Language: English
• Published: England 26.03.2019
• Subjects: Clonogenic assays; Genomic editing; Cell clones; Signaling cascades
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.217661

Genomic editing methods, such as the CRISPR/Cas9 system, are routinely used to study gene function in somatic cells. Owing to the heterogeneity of mutations, it is necessary to purify cell clones grown from high dilution to the point of colony formation, which can be a time-consuming process. Here, we tested a modified approach in which we seeded cells at high dilution, together with non-edited carrier cells. As a comparison, cells were also grown at high dilution with conditioned medium from a high-density culture. When using carrier cells or conditioned medium, the formation of cell colonies is accelerated. Additionally, clones grown with carrier cells are more similar to the parental lines in terms of their tumorigenic properties. Surprisingly, key signaling cascades are highly divergent between clones isolated from low-density cultures, even with conditioned medium, in contrast to clones isolated with carrier cells. Thus, our study uncovers a significant limitation using the common approach of isolating cell clones following genetic modifications and suggests an alternative method that mitigates the problem of heterogeneity of gene expression between clones.This article has an associated First Person interview with the first author of the paper.