Abstract
A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, because temporal changes in intracellular protein abundances cannot be measured directly in live cells, fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systematically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog, we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight the problem of measurement in live cells.
Highlights
Fluorescence has been used to report expression of gene products in live cells since green fluorescent protein (GFP) was first cloned and utilized as a tracer [1,2]
For such constructs to be effective at the single cell level, the fluorescence signal driven from the reporter allele should accurately represent protein expression from the wild-type allele
We will focus on mRNA dynamics, but similar reasoning may be extended to the protein level, and the general conclusions that we draw are not limited to heterozygous reporters
Summary
Fluorescence has been used to report expression of gene products in live cells since green fluorescent protein (GFP) was first cloned and utilized as a tracer [1,2]. Live cell fluorescence imaging and analysis techniques allow investigation of temporal changes in protein expression and have become an essential tool in modern molecular biology [3]. Their proper use requires the reporter signal to be representative of expression of the protein of interest at the scale of interest. If the reporter is to be used as a proxy for protein expression within a single cell, to be able to draw accurate conclusions, the reporter signal should be representative of protein expression in that particular cell. The ways in which the genetic manipulations involved in generating reporter cell lines affect endogenous gene expression kinetics are not well understood
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