Single-molecule techniques to probe the dynamic gene regulatory network formed by core pluripotency circuit in embryonic stem cells

• Main Author: Lin, Ya
• Other Authors: van Oudenaarden, Alexander
• Language: en_US
• Published: Harvard University 2014
• Subjects: Biophysics
• Online Access: http://dissertations.umi.com/gsas.harvard.inactive:11737; http://nrs.harvard.edu/urn-3:HUL.InstRepos:13070085

This work investigates the dynamics of gene regulatory network formed by Oct4, Sox2 and Nanog in embryonic stem cells (ESCs). Despite a large number of existing studies on stem cells, current technologies used often force a compromise between quantification of gene expression via bulk measurements and qualitative imaging of cell heterogeneity. There are few options that allow for accurate and quantitative single-cell analysis that is robust yet not associated with a high degree of technical difficulty or obscured by amplification. Here, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression of the core pluripotency circuit upon various types of perturbations such as differentiation, induction or knockdown of one of the three pluripotent factors. We used previously-published smFISH procedures as our initial template for investigating gene regulatory dynamics of the core pluripotency circuit during those perturbation assays. To obtain a more comprehensive picture of the regulatory circuit, we developed a modified smFISH strategy to measure mRNA and protein expression simultaneously in single ESCs. By incorporating a novel modification into the smFISH technique which allows accurate quantification of transcripts that differ by short sequences, we managed to identify a few interesting features of the core pluripotency circuit. Taken together, we demonstrated our ability to perform single-cell, single-molecule assays that reveal highly quantitative information in unprecedented detail.