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Methods and Sensors For Functional Genomic Studies of Cell‐Cycle Transitions in Single Cells
Author(s) -
Zambon Alex,
Hsu Tom,
Klinck Miranda,
Patam Leomar,
Hickerson Anna
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06082
Subject(s) - cell cycle , population , biology , cell division , cell , single cell analysis , microbiology and biotechnology , computational biology , stem cell , genetics , demography , sociology
Our understanding of the genetic control mechanisms governing the cell cycle has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our fundamental understanding of much of what we know about how the cell cycle is regulated, these approaches also mask the signaling and genetic signatures of rare subpopulations. Quiescent (G 0 ) or very slow dividing (SD) cells are two such related populations. Our data and that of others show that the majority of single clonal MCF7 breast cancer cells cultured at subconfluency can complete a full round of cell division in as little as 24h, however subpopulations within that culture (1–5%) can require over 100h to complete the cell cycle. Moreover, it is not clear how these phenotypes are transferred to daughter cells. Understanding the proteins and networks that control G 0 or SD cells may lead to the identification of new drug targets for aging or tissue regeneration. This study establishes an integrated computational, imaging and molecular pipeline that enables the phenotyping of G 0 or SD cultured cells within a population. We define imaging parameters and open source R scripts to generate and analyze time‐lapse images to track and quantify cell cycle transition in individual cells expressing a genetically encoded cell cycle reporter system (Ki67p‐FUCCI). We report how these cells can be used to identify transcriptomic signatures of G 0 , G 1 and G 2 phase cells within a population. Additionally, we present single‐cell microinjection parameters to optimize single cell transfection by direct injection of mRNA in MCF7 cells with detectable reporter gene expression (GFP) within 5h of microinjection. These findings coupled with the possibility of targeting several hundreds of single cells within a field of view while simultaneously conducting 4 channel time‐lapse microscopy dramatically increase throughput and sensitivity over conventional methods for functional genomics studies to identify new genes that control cell cycle transitions.