Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

Mineral Composition is Altered by Osteoblast Expression of an Engineered Gs-Coupled Receptor

Activation of the Gs G protein–coupled receptor Rs1 in osteoblasts increases bone mineral density by 5- to 15-fold in mice and recapitulates histologic aspects of fibrous dysplasia of the bone. However, the effects of constitutive Gs signaling on bone tissue quality are not known. The goal of this study was to determine bone tissue quality in mice resulting from osteoblast-specific constitutive Gs activation, by the complementary techniques of FTIR spectroscopy and synchrotron radiation micro-computed tomography (SRμCT). Col1(2.3)-tTA/TetO-Rs1 double transgenic (DT) mice, which showed osteoblast-specific constitutive Gs signaling activity by the Rs1 receptor, were created. Femora and calvariae of DT and wild-type (WT) mice (6 and 15 weeks old) were analyzed by FTIR spectroscopy. WT and DT femora (3 and 9 weeks old) were imaged by SRμCT. Mineral-to-matrix ratio was 25% lower (P = 0.010), carbonate-to-phosphate ratio was 20% higher (P = 0.025), crystallinity was 4% lower (P = 0.004), and cross-link ratio was 11% lower (P = 0.025) in 6-week DT bone. Differences persisted in 15-week animals. Quantitative SRμCT analysis revealed substantial differences in mean values and heterogeneity of tissue mineral density (TMD). TMD values were 1,156 ± 100 and 711 ± 251 mg/cm3 (mean ± SD) in WT and DT femoral diaphyses, respectively, at 3 weeks. Similar differences were found in 9-week animals. These results demonstrate that continuous Gs activation in murine osteoblasts leads to deposition of immature bone tissue with reduced mineralization. Our findings suggest that bone tissue quality may be an important contributor to increased fracture risk in fibrous dysplasia patients

Rapid measurement of intravoxel incoherent motion (IVIM) derived perfusion fraction for clinical magnetic resonance imaging

Objective This study aimed to investigate the reliability of intravoxel incoherent motion (IVIM) model derived parameters D and f and their dependence on b value distributions with a rapid three b value acquisition protocol. Materials and methods Diffusion models for brain, kidney, and liver were assessed for bias, error, and reproducibility for the estimated IVIM parameters using b values 0 and 1000, and a b value between 200 and 900, at signal-to-noise ratios (SNR) 40, 55, and 80. Relative errors were used to estimate optimal b value distributions for each tissue scenario. Sixteen volunteers underwent brain DW-MRI, for which bias and coefficient of variation were determined in the grey matter. Results Bias had a large influence in the estimation of D and f for the low-perfused brain model, particularly at lower b values, with the same trends being confirmed by in vivo imaging. Significant differences were demonstrated in vivo for estimation of D (P = 0.029) and f (P < 0.001) with [300,1000] and [500,1000] distributions. The effect of bias was considerably lower for the high-perfused models. The optimal b value distributions were estimated to be brain500,1000, kidney300,1000, and liver200,1000. Conclusion IVIM parameters can be estimated using a rapid DW-MRI protocol, where the optimal b value distribution depends on tissue characteristics and compromise between bias and variability

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field

Gene Expression Profiles of the NCI-60 Human Tumor Cell Lines Define Molecular Interaction Networks Governing Cell Migration Processes

Although there is extensive information on gene expression and molecular interactions in various cell types, integrating those data in a functionally coherent manner remains challenging. This study explores the premise that genes whose expression at the mRNA level is correlated over diverse cell lines are likely to function together in a network of molecular interactions. We previously derived expression-correlated gene clusters from the database of the NCI-60 human tumor cell lines and associated each cluster with function categories of the Gene Ontology (GO) database. From a cluster rich in genes associated with GO categories related to cell migration, we extracted 15 genes that were highly cross-correlated; prominent among them were RRAS, AXL, ADAM9, FN14, and integrin-beta1. We then used those 15 genes as bait to identify other correlated genes in the NCI-60 database. A survey of current literature disclosed, not only that many of the expression-correlated genes engaged in molecular interactions related to migration, invasion, and metastasis, but that highly cross-correlated subsets of those genes engaged in specific cell migration processes. We assembled this information in molecular interaction maps (MIMs) that depict networks governing 3 cell migration processes: degradation of extracellular matrix, production of transient focal complexes at the leading edge of the cell, and retraction of the rear part of the cell. Also depicted are interactions controlling the release and effects of calcium ions, which may regulate migration in a spaciotemporal manner in the cell. The MIMs and associated text comprise a detailed and integrated summary of what is currently known or surmised about the role of the expression cross-correlated genes in molecular networks governing those processes