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1.
Nucleic Acids Res ; 42(21): 13440-51, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25378321

ABSTRACT

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.


Subject(s)
Cell Engineering/methods , Gene Regulatory Networks , Animals , Cell Line , Cloning, Molecular , Gene Library , Humans
2.
Mol Cell Proteomics ; 12(12): 3704-18, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24023389

ABSTRACT

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.


Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Platelet Factor 4/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/metabolism , Cell Movement , Collagen/chemistry , Dermis/blood supply , Dermis/cytology , Dermis/drug effects , Dermis/metabolism , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Gels , Gene Expression Regulation , Humans , Mass Spectrometry , Molecular Sequence Annotation , Neovascularization, Physiologic , Phosphotyrosine/metabolism , Platelet Factor 4/genetics , Platelet Factor 4/pharmacology , Primary Cell Culture , Protein Binding , Receptor, EphA2/genetics , Receptor, EphA2/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacology , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolism
3.
Integr Biol (Camb) ; 5(3): 510-22, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23303249

ABSTRACT

Angiogenesis requires coordinated dynamic regulation of multiple phenotypic behaviors of endothelial cells in response to environmental cues. Multi-scale computational models of angiogenesis can be useful for analyzing effects of cell behaviors on the tissue level outcome, but these models require more intensive experimental studies dedicated to determining the required quantitative "rules" for cell-level phenotypic responses across a landscape of pro- and anti-angiogenic stimuli in order to ascertain how changes in these single cell responses lead to emerging multi-cellular behavior such as sprout formation. Here we employ single-cell microscopy to ascertain phenotypic behaviors of more than 800 human microvascular endothelial cells under various combinational angiogenic (VEGF) and angiostatic (PF4) cytokine treatments, analyzing their dynamic behavioral transitions among sessile, migratory, proliferative, and apoptotic states. We find that an endothelial cell population clusters into an identifiable set of a few distinct phenotypic state transition patterns (clusters) that is consistent across all cytokine conditions. Varying the cytokine conditions, such as VEGF and PF4 combinations here, modulates the proportion of the population following a particular pattern (referred to as phenotypic cluster weights) without altering the transition dynamics within the patterns. We then map the phenotypic cluster weights to quantified population level sprout densities using a multi-variate regression approach, and identify linear combinations of the phenotypic cluster weights that associate with greater or lesser sprout density across the various treatment conditions. VEGF-dominant cytokine combinations yielding high sprout densities are characterized by high proliferative and low apoptotic cluster weights, whereas PF4-dominant conditions yielding low sprout densities are characterized by low proliferative and high apoptotic cluster weights. Migratory cluster weights show only mild association with sprout density outcomes under the VEGF/PF4 conditions and the sprout formation characteristics explored here.


Subject(s)
Cytokines/metabolism , Endothelial Cells/cytology , Neovascularization, Pathologic , Algorithms , Apoptosis , Cell Culture Techniques , Cell Movement , Cell Proliferation , Cluster Analysis , Collagen/metabolism , Endothelium, Vascular/metabolism , Humans , Microcirculation , Models, Biological , Multivariate Analysis , Phenotype , Regression Analysis
4.
PLoS One ; 7(12): e50582, 2012.
Article in English | MEDLINE | ID: mdl-23226527

ABSTRACT

In recent years, microfluidic systems have been used to study fundamental aspects of angiogenesis through the patterning of single-layered, linear or geometric vascular channels. In vivo, however, capillaries exist in complex, three-dimensional (3D) networks, and angiogenic sprouting occurs with a degree of unpredictability in all x,y,z planes. The ability to generate capillary beds in vitro that can support thick, biological tissues remains a key challenge to the regeneration of vital organs. Here, we report the engineering of 3D capillary beds in an in vitro microfluidic platform that is comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads. The engineered vessels have patent lumens, form robust ~1.5 mm capillary networks across the devices, and support the perfusion of 1 µm fluorescent beads through them. In addition, the alginate beads offer a modular method to encapsulate and co-culture cells that either promote angiogenesis or require perfusion for cell viability in engineered tissue constructs. This laboratory-constructed vascular supply may be clinically significant for the engineering of capillary beds and higher order biological tissues in a scalable and modular manner.


Subject(s)
Capillaries/growth & development , Neovascularization, Physiologic , Alginates , Cells, Cultured , Coculture Techniques , Glucuronic Acid , Hexuronic Acids , Humans , Immunohistochemistry , Microfluidics , Tissue Scaffolds
7.
J Biomol Screen ; 12(5): 683-93, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17507638

ABSTRACT

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.


Subject(s)
Biological Assay/methods , Cadherins/analysis , Epithelial Cells/physiology , Proteins/metabolism , beta Catenin/analysis , Animals , Antibodies, Monoclonal/metabolism , Breast Neoplasms/pathology , Cadherins/metabolism , Cadherins/physiology , Cell Adhesion , Cell Line , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Female , Humans , Kidney/cytology , Mice , Plasmids , Reproducibility of Results , Retroviridae/genetics , Transfection , beta Catenin/metabolism , beta Catenin/physiology
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