Kettering University

About: Kettering University is a education organization based out in Flint, Michigan, United States. It is known for research contribution in the topics: RNA & Antigen. The organization has 6842 authors who have published 7689 publications receiving 337503 citations. The organization is also known as: GMI Engineering & Management Institute & General Motors Institute.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish

Abstract: Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, μ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

Method for simple analysis of relative nucleic acid levels in multiple small samples by cytoplasmic dot hybridization

Abstract: ? 280 HRL/JEL Abstract A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numberous small samples of biological specimens is described The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis are minimized Experiments with a clonal line of rat pituitary tumor (GH3) cells show that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 25 x 104 cells The specificity of the technique for prolactin mRNA is shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences Experiments with cultured rat hemipituitaries show that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary can be readily detected This technique is quite simple, can be quantified, and permits the ? 280 HRL/JEL simultaneous analysis of multiple samples while requiring very small amounts of material for analysis Hence, it should be quite useful for example for studies with various experimental systems of the regulation of specific mRNA levels

Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching.

Abstract: Dynamic cell movements and rearrangements are essential for the generation of the mammalian body plan, although relatively little is known about the genes that coordinate cell movement and cell fate. WAVE complexes are regulators of the actin cytoskeleton that couple extracellular signals to polarized cell movement. Here, we show that mouse embryos that lack Nap1, a regulatory component of the WAVE complex, arrest at midgestation and have defects in morphogenesis of all three embryonic germ layers. WAVE protein is not detectable in Nap1 mutants, and other components of the WAVE complex fail to localize to the surface of Nap1 mutant cells; thus loss of Nap1 appears to inactivate the WAVE complex in vivo. Nap1 mutants show specific morphogenetic defects: they fail to close the neural tube, fail to form a single heart tube (cardia bifida), and show delayed migration of endoderm and mesoderm. Other morphogenetic processes appear to proceed normally in the absence of Nap1/WAVE activity: the notochord, the layers of the heart, and the epithelial-to-mesenchymal transition (EMT) at gastrulation appear normal. A striking phenotype seen in approximately one quarter of Nap1 mutants is the duplication of the anteroposterior body axis. The axis duplications arise because Nap1 is required for the normal polarization and migration of cells of the Anterior Visceral Endoderm (AVE), an early extraembryonic organizer tissue. Thus, the Nap1 mutant phenotypes define the crucial roles of Nap1/WAVE-mediated actin regulation in tissue organization and establishment of the body plan of the mammalian embryo.