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）：父母补助金（前胶原蛋白C-蛋白酶增强剂：本修订申请的体内作用，R 01 AR 53815）涉及PCOLCE 1-null，PCOLCE 2-null，和PCOLCE 1/PCOLCE 2双无效基因敲除小鼠，以研究两种前胶原C蛋白酶增强蛋白PCOLCE 1和PCOLCE 2的体内作用，所述蛋白增强蛋白PCOLCE 1和PCOLCE 2增强BMP 1 - 2的能力。如细胞外蛋白酶从主要的纤维原胶原中生物合成地切割C-前肽。母基金是主要研究者以前赠款的延续，主要研究BMP 1样蛋白酶、其底物、生物学功能和这些功能的调节剂。然而，目前的母基金并没有调查BMP 1样蛋白酶的其他生物学作用，而不是受PCOLCE 1和2的影响。在这里，我们提出了BMP 1样蛋白酶的其他功能的直接研究，这将通过使用本实验室创建的新试剂和我们以前的研究中获得的技能来利用。具体地说，尽管Tll 1基因（编码BMP 1样蛋白酶，哺乳动物tolloid-like 1（mTLL 1））的敲除是胚胎致死的，但我们最近成功地创建了具有floxed Tll 1等位基因的小鼠，这将允许组织特异性Tll 1敲除。我们已经证明，Tll 1在发育和成人中枢神经系统（CNS）的特定结构中高度表达。因此，我们已经将我们的floxed Tll 1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杂交，并在本文中证明了Tll 1的CNS特异性敲除。我们建议使用这些小鼠来确定体内Tll 1 CNS功能。我们要求为实验室成员提供工资支持，以进行此类研究。此外，我们已经开始使用强大的斑马鱼系统来进一步定义BMP 1样金属蛋白酶的功能。然而，我们以前在这个系统中的工作使用了另一个实验室的设备和鱼，大大延迟了我们的研究。我们建议购买设备，使我们能够在我们的实验室进行斑马鱼的工作。我们建议首先使用这样的设备，以确定在体内的作用的非特征蛋白质斑马鱼类软骨（zCHL），我们已经发现这是一个母亲的因素在最早的胚胎，并假设是一个基板的BMP 1样蛋白酶，参与调节BMP信号。获得该设备不仅将大大提高成功完成zCHL研究的能力，而且将大大提高我们使用强大的斑马鱼系统进一步探索BMP 1样蛋白酶和ECM组分的能力，这些蛋白酶和ECM组分也是本实验室的研究重点。 公共卫生相关性：我们发现，移除小鼠体内的一个特定基因会导致心脏的位置错误，而且不能很好地维持胚胎的存活。我们还发现，该基因在发育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，只在中枢神经系统中复制这种基因，这样做的动物不会在胚胎阶段死于心脏缺陷，而是会发育成成年人，我们可以研究这种基因在大脑结构中的损失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，来研究我们发现的基因在早期发育中的作用。我们认为这种蛋白质在决定胚胎的前部和后部方面很重要。这项研究选择了斑马鱼，因为它们产生的卵子在外部受精并在母体外发育。因此，胚胎可以在发育的所有阶段进行观察和遗传操作，而不需要对母亲进行任何侵入性程序。斑马鱼胚胎是光学透明的，发育迅速，允许人们分析发育事件，因为它们发生在完整的胚胎。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，从而了解这些基因中的缺陷如何参与人类疾病。