4 research outputs found
Design and Validate a Synthetic Circuit for Detecting Pathway Signaling in Mammalian Cells
Synthetic circuits provide novel ways for scientists to program and probe mammalian cell behavior. This allows for enhanced research tools and chemical sensors. Our project works with an engineered synthetic circuit and monitors the output of the circuit using green fluorescent protein( GFP) and luciferase. Expression of GFP is both quantifiable and observable over a time period in living cells. Luciferase can be detected by an assay in lysed cells to give data on overall expression of the circuit. By measuring and comparing output of this circuit over time, we can create a model to demonstrate the lack of expression, minimal expression, and maximum expression of the circuit. Our dual reporter system can be adapted to give detailed information on various promoter elements of synthetic circuits.
This dual reporter system was designed to monitor s ignal pathways within mammalian cells. Signal pathways are a complex and dynamic interconnected web of protein expression. Methods to monitor these pathways en vivo poses a challenge due to lack of noninvasive reporting systems in desired cell models. The use of a dualreporter system with expression of GFP and firefly luciferase allows for the assessment of signal transduction with resulting data sets of overall intensity and temporality in realtime. Neither light emitting marker could show these two properties alone. To demonstrate this system and establish a scale of expression, we transformed mammalian HEK cells with a vector coding for the dual reporter. The transformed cell lines were monitored via fluorescent microscopy and luciferase assays. Our findings show successful basal level expression of synthetic circuit dualreporter system both qualitatively and quantitatively in a way that neither reporter protein could achieve alone. Our system can successfully deliver this data type and can be an effective tool for cell signal pathway expression
Integrated Single-Cell Transcriptomics and Chromatin Accessibility Analysis Reveals Novel Regulators of Mammary Epithelial Cell Identity
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Integrated Single-Cell Transcriptomics and Chromatin Accessibility Analysis Reveals Regulators of Mammary Epithelial Cell Identity
The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer