A Cre/loxP binary genetic approach to study murine spinal cord and limb development.
Item
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Title
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A Cre/loxP binary genetic approach to study murine spinal cord and limb development.
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Identifier
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AAI9820554
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identifier
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9820554
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Creator
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Li, Xue.
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Contributor
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Adviser: Thomas Lufkin
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Date
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1998
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Neuroscience
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Abstract
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The Cre/loxP site-specific recombination system was designed to conditionally express a battery of dorsal neural tube specific genes in the ventral neural tube to investigate their function in neural tube patterning. Two different types of transgenic mice, E3Cre and E3loxP, were generated using the same promoter which has specific activities in the floor plate and notochord between embryonic day 8.0-14.5. The E3Cre transgene encodes Cre and the alkaline phosphatase reporter gene, while the E3loxP transgene encodes a STOP sequence flanked by loxP sites, which prevents translation of 3{dollar}\sp\prime{dollar} cDNAs. Double transgenic embryos harboring both E3Cre and E3loxP transgenes display specific excision of the STOP sequence demonstrating that Cre-Mediated recombination occurs efficiently in vivo. However, expression of downstream cDNAs is not detected. Unexpectedly, elevated levels of specific cell death in the floor plate and notochord is apparent and is independent of the 3{dollar}\sp\prime{dollar} cDNAs included in the E3loxP transgene expression cassette.;In concert with the discovery that Cre-mediated recombination leads to chromosome deletion in transgenic mice, molecular studies in the double transgenic embryos suggest that the Cre-mediated recombination directly causes apoptosis of specific cells. Molecular and immunohistochemical analyses demonstrate that the floor plate and notochord play important roles for differentiation of the motor neurons, and for correct projection patterning of commissural axons. Strikingly, forelimbs fail to develop normally in the double transgenic embryos. This morphological defect is apparent at E9.5, at the time when forelimb buds normally appear from the flanking regions. Molecular analyses demonstrate that the earliest known limb inducing molecules, including fibroblast growth factors 8 and 10, are not expressed in the defective forelimbs, suggesting that the defect is due to a failure of limb initiation. In view of these findings, we propose that putative limb inducing signals are present transiently in axial structures, the node, notochord or floor plate. The inducing signals are then propagated through paraxial, intermediate, lateral plate mesoderm and finally to the surface ectoderm to form the limb bud. Once the limb bud is formed, its outgrowth and patterning continue in a self-organizing fashion.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
