Koyano-Nakagawa N.,University of Minnesota |
Kweon J.,University of Minnesota |
Iacovino M.,University of Minnesota |
Shi X.,University of Minnesota |
And 7 more authors.
Stem Cells | Year: 2012
During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2+, c-kit+, and CD41+ hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP1 population. In the Etv2 mutant yolk sac, Tie2+ cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2. © AlphaMed Press.
Zhou X.,LC science LLC |
Zhu Q.,LC science LLC |
Eicken C.,LC science LLC |
Sheng N.,Atactic Technologies Inc. |
And 3 more authors.
Methods in Molecular Biology | Year: 2012
The diverse functions of microRNA (miRNA) molecules have drawn broad and intensive interest in various biological fields, biomedical applications, and technology development. Which are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (1). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (2-4). miRNAs are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (Bartel, Cell 116:281-97, 2004). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (He et al., Nature 435:828-833, 2005; Lu et al., Nature 435:834-838, 2005; O'Donnell, et al., Nature 435:839-843, 2005). In this chapter, we describe the methods using μParaflo ® microfluidic oligonucleotide microarray technology for applications in miRNA profiling. One unique feature of this technology is the flexibility that provides users with the freedom to select sequence content either for focused studies wherein only the most relevant sequences are included or for discovery studies wherein the most updated sequence content such as those newly derived from deep sequencing. This chapter provides detailed information from experimental design to sample preparation, as well as data analysis for a miRNA array experiment. © 2012 Springer Science+Business Media, LLC.
Lukiw W.J.,Louisiana State University Health Sciences Center |
Dua P.,Louisiana Technical University |
Pogue A.I.,Alchem Biotek Corporation |
Eicken C.,LC science Corporation |
Hill J.M.,Louisiana State University Health Sciences Center
Journal of Toxicology and Environmental Health - Part A: Current Issues | Year: 2011
A mouse- and human-brain-abundant, nuclear factor (NF)-kB-regulated, micro RNA-146a (miRNA-146a) is an important modulator of the innate immune response and inflammatory signaling in specific immunological and brain cell types. Levels of miRNA-146a are induced in human brain cells challenged with at least five different species of single- or double-stranded DNA or RNA neurotrophic viruses, suggesting a broad role for miRNA-146a in the brain's innate immune response and antiviral immunity. Upregulated miRNA-146a is also observed in pro-inflammatory cytokine-, Aβ42 peptide- and neurotoxic metal-induced, oxidatively stressed human neuronal-glial primary cell cocultures, in murine scrapie and in Alzheimer's disease (AD) brain. In AD, miRNA-146a levels are found to progressively increase with disease severity and co-localize to brain regions enriched in inflammatory neuropathology. This study provides evidence of upregulation of miRNA-146a in extremely rare (incidence 1-10 per 100 million) human prion-based neurodegenerative disorders, including sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). The findings suggest that an upregulated miRNA-146a may be integral to innate immune or inflammatory brain cell responses in prion-mediated infections and to progressive and irreversible neurodegeneration of both the murine and human brain. Copyright © Taylor & Francis Group, LLC.
Jin Y.-F.,Zhejiang Sci-Tech University |
Xu G.-M.,LC Science Co. |
Li Y.,Zhejiang Sci-Tech University |
Meng L.,Zhejiang Sci-Tech University |
And 3 more authors.
Progress in Biochemistry and Biophysics | Year: 2011
Data normalization plays a crucial role in the interpretation of experimental result. House-keeping genes were utilized as internal controls to accurately determine the gene expression in quantitative PCR. However, significant expression variation of these internal controls was revealed recently. A novel normalization approach (per cell normalization, percellome), which is based on DNA and RNA normalization, is developed to calibrate miRNA expression in quantitative PCR. In which, a cocktail of three external RNAs with different copy numbers were spiked, so as be able to normalize miRNA expression against cell number. Gene expression of 14 miRNAs, as well as commonly used internal controls (U6 ncRNA and 5S rRNA), were examined in the brain samples of 8 and 40 week-old mice. By using "per cell normalization" method, the expression level of theses miRNAs varied from 2- to 26-fold, while the absolute miRNA copy number per cell were from 2.0×10 5 to 4.3 × 10 5 copies per cell. Interestingly, the fold-change of U6 ncRNA and 5S rRNA were found to be 1.5- and 4.8-fold (based on DNA normalization), and 5.8- and 3.8-fold (based on RNA normalization), indicating significant expression variations of these two house-keeping genes. The study provides an novel approach to reliably normalize miRNA expression in quantitative PCR.
LC science LLC | Date: 2015-04-27
Reagents for scientific and or medical research use, namely, reagents for use in preparation of DNA sequencing samples.
LC science LLC | Date: 2015-04-27
Microfluidic chips in the nature of chemical, biochemical and/or biological assays and reagents for use in biological and biochemical research.