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Living gene regulation

Context.  Understanding cellular differentiation is one of the main challenges facing modern biomedical research. Most models of cellular differentiation equate this process with the establishment of a unique and irreversible gene expression program. In recent years however, numerous studies have shown that differentiated cells could be re-programmed in vitro to stem cell-like cells, albeit at low efficiency. How this is achieved, particularly at the level of gene expression, remains to be elucidated. The possibility of re-programming cells implicates a certain degree of plasticity in gene expression programs. We postulate that this plasticity is due to transcriptional pulsing [1], a recently-discovered phenomenon whereby RNA synthesis occurs in bursts instead of being continuous.

Goal.  To image transcription of silenced and activated genes during differentiation of mouse embryonic stem cells along the mesodermic lineage and to measure changes, if any, in the frequency of transcriptional pulsing during this process.

Methodology.  Our laboratory uses the MS2-GFP reporter system to image living transcription [2]. In this system, first developed by the Singer lab, an heterologous MS2 sequence is engineered into the transcribed portion of a gene of interest and the chimaeric RNA is detected in living cells through binding of a MS2-binding fluorescent protein.