PROCOLLAGEN C-PROTEINASE ENHANCERS: IN VIVO ROLES

原胶原 C-蛋白酶增强剂：体内作用

• 批准号: 7811594
• 负责人: DANIEL S GREENSPAN
• 金额: $ 41.64万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-24 至 2011-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811594
• 关键词: Ablation; Adult; Affect; Alleles; Animals; Back; Behavioral; Biological; Biological Assay; Biological Process; Brain; Cleaved cell; Congenital Heart Defects; Defect; Development; Embryo; Enhancers; Equipment; Event; Extracellular Matrix; Fishes; Genes; Grant; Heart; Hippocampus (Brain); Human; In Situ Hybridization; In Vitro; Knock-out; Knockout Mice; Laboratories; Metalloproteases; Methods; Mothers; Mus; Nervous System Physiology; Neuraxis; Peptide Hydrolases; Physiological; Principal Investigator; Procedures; Procollagen; Proteins; RNA; RNA Splicing; Reagent; Research; Role; Signal Transduction; Site; Staging; Structure; System; Testing; Tissues; Untranslated Regions; Wages; Work; Zebrafish; Zebrafish Proteins; behavior test; chordin; extracellular; genetic manipulation; human disease; in vivo; insight; knock-down; loss of function; member; nestin protein; novel; overexpression; parent grant; procollagen C-endopeptidase; public health relevance; recombinase; research study; skills; zygote

DESCRIPTION (provided by applicant): The parent grant (Procollagen C-proteinase Enhancers: In vivo Roles, R01 AR53815) for this Revision Application involves in depth characterization of PCOLCE1-null, PCOLCE2-null, and PCOLCE1/PCOLCE2 doubly null knockout mice to study in vivo roles of the two procollagen C-proteinase enhancer proteins PCOLCE1 and PCOLCE2 which enhance the ability of BMP1-like extracellular proteinases to biosynthetically cleave the C-propeptides from the major fibrillar procollagens. The parent grant is a continuation of work from previous grants of the principal investigator that focused on the BMP1-like proteinases, their substrates, biological functions, and modulators of those functions. However, the present parent grant does not investigate additional biological roles of BMP1-like proteinases, other than those affected by PCOLCE1 and 2. Here we propose direct studies of additional functions of the BMP1-like proteinase, that will be leveraged by use of novel reagents created in this lab, and skills obtained in our previous studies. Specifically, although knockout of the Tll1 gene, which encodes the BMP1-like proteinase, mammalian tolloid-like 1 (mTLL1), is embryonic lethal, we have recently been successful in creating mice with floxed Tll1 alleles, which will allow tissue-specific Tll1 knockout. We've shown that Tll1 is highly expressed in specific structures of the developing and adult central nervous system (CNS). Thus, we've crossed our floxed Tll1 mice to mice in which Cre recombinase is driven by the CNS-specific enhancer sequences of the nestin gene, and demonstrate herein CNS-specific knockout of Tll1. We propose using these mice to determine in vivo Tll1 CNS functions. We request salary support for a lab member to conduct such studies. In addition, we have begun using the powerful zebrafish system to further define functions of the BMP1-like metalloproteinases. However, our previous work in this system employed the equipment and fish of another lab, greatly delaying our studies. We propose acquiring equipment that will enable us to perform zebrafish work in our lab. We propose first using such equipment to determine in vivo roles of the uncharacterized protein zebrafish Chordin-like (zCHL), which we have found to be a maternal factor in earliest embryos, and hypothesize to be a substrate of BMP1-like proteinases, involved in regulating BMP signaling. Obtaining the equipment will not only greatly enhance ability to successfully complete the zCHL study, but will greatly enhance our ability to use the powerful zebrafish system to further explore the BMP1-like proteinases, and ECM components that have also been the focus of research in this laboratory. PUBLIC HEALTH RELEVANCE: We've found that removing a particular gene in mice results in hearts that are in the wrong place, and which don't work sufficiently well to keep the embryo alive. We also have found that this gene is expressed at high levels in the developing and adult central nervous system. We propose using a method that will inactivate this gene only in the central nervous system, so that animals in which this is done will not die of heart defects in the embryonic stage, but will develop into adults in which we can study the effects of loss of this gene in the structure of the brain and its functioning in behavioral and physiological tests. In addition, we propose using a small tropical fish, known as the zebrafish, to examine the role in early development of a gene we've discovered. We believe that this protein is important in determining what will be the front and what will be the back of the embryo. Zebrafish were chosen for this study, as they generate eggs that are fertilized externally and develop outside the mother. The embryos are therefore accessible for observation and genetic manipulation at all stages of development without requiring any invasive procedures on the mother. Zebrafish embryos are optically transparent and develop rapidly, allowing one to analyze developmental events as they occur in the intact embryo. We believe that insights obtained from study in the mouse and zebrafish systems will provide important insights into the functions of the related human genes, and thus into how defects in such genes might be involved in human disease.

描述（由申请人提供）：本修订申请的母基金（原胶原 C 蛋白酶增强剂：体内作用，R01 AR53815）涉及对 PCOLCE1 无效、PCOLCE2 无效和 PCOLCE1/PCOLCE2 双无效敲除小鼠的深入表征，以研究两种前胶原 C 蛋白酶增强蛋白 PCOLCE1 和 PCOLCE2 的体内作用，它们增强了BMP1 样细胞外蛋白酶以生物合成方式从主要纤维状前胶原中裂解 C 前肽。母基金是主要研究人员先前资助工作的延续，重点关注 BMP1 样蛋白酶、其底物、生物功能以及这些功能的调节剂。然而，目前的家长资助并没有研究 BMP1 样蛋白酶的其他生物学作用，除了受 PCOLCE1 和 2 影响的作用之外。在这里，我们建议直接研究 BMP1 样蛋白酶的其他功能，这将通过使用新型本实验室创建的试剂以及我们之前的研究中获得的技能。具体来说，虽然编码 BMP1 样蛋白酶、哺乳动物 tollloid-like 1 (mTLL1) 的 Tll1 基因的敲除是胚胎致死的，但我们最近成功地培育出了具有 floxed Tll1 等位基因的小鼠，这将允许组织特异性 Tll1昏死。我们已经证明 Tll1 在发育中和成人中枢神经系统 (CNS) 的特定结构中高度表达。因此，我们将 floxed Tll1 小鼠与 Cre 重组酶由巢蛋白基因的 CNS 特异性增强子序列驱动的小鼠进行杂交，并在本文中证明了 Tll1 的 CNS 特异性敲除。我们建议使用这些小鼠来确定体内 Tll1 CNS 功能。我们要求为进行此类研究的实验室成员提供工资支持。此外，我们已经开始使用强大的斑马鱼系统来进一步定义 BMP1 样金属蛋白酶的功能。然而，我们之前在该系统中的工作使用了另一个实验室的设备和鱼，极大地延误了我们的研究。我们建议购买设备，使我们能够在实验室中进行斑马鱼工作。我们建议首先使用此类设备来确定未表征的斑马鱼 Chordin 样蛋白 (zCHL) 的体内作用，我们发现它是最早胚胎中的母体因素，并假设它是 BMP1 样蛋白酶的底物，参与调节 BMP 信号传导。获得该设备不仅将大大增强顺利完成zCHL研究的能力，也将大大增强我们利用强大的斑马鱼系统进一步探索本领域研究重点的类BMP1蛋白酶和ECM成分的能力。实验室。 公共健康相关性：我们发现，去除小鼠体内的特定基因会导致心脏处于错误的位置，并且无法充分发挥作用以保持胚胎存活。我们还发现该基因在发育中和成人中枢神经系统中高水平表达。我们建议使用一种方法，仅在中枢神经系统中灭活该基因，这样，这样做的动物就不会在胚胎阶段因心脏缺陷而死亡，而是会发育成成年人，我们可以在其中研究损失的影响。该基因在大脑结构中的作用及其在行为和生理测试中的功能。此外，我们建议使用一种小型热带鱼（称为斑马鱼）来检查我们发现的基因在早期发育中的作用。我们相信这种蛋白质对于确定胚胎的正面和背面非常重要。斑马鱼被选用于这项研究，因为它们产生的卵在体外受精并在母体体外发育。因此，胚胎在发育的各个阶段都可以进行观察和基因操作，而不需要对母亲进行任何侵入性手术。斑马鱼胚胎是光学透明的并且发育迅速，使得人们能够分析完整胚胎中发生的发育事件。我们相信，从小鼠和斑马鱼系统的研究中获得的见解将为相关人类基因的功能提供重要的见解，从而了解这些基因的缺陷如何与人类疾病有关。