CALCITONIN RECEPTORS IN DEVELOPMENT

降钙素受体的发育

• 批准号: 6100314
• 负责人: STEVEN GOLDRING
• 金额: --
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6100314
• 关键词: autoradiography; calcitonin; early embryonic stage; embryonic stem cell; genetic transcription; genetically modified animals; hormone receptor; immunocytochemistry; in situ hybridization; laboratory mouse; microinjections; molecular cloning; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein structure function; receptor expression; tissue /cell culture; vertebrate embryology; zebrafish

Calcitonin (CT) was originally identified as a peptide hormone released by parafollicular cells of the thyroid gland in response to hypercalcemic stimuli. In mammals, it was found that CT secretion produces a rapid lowering of extracellular calcium levels by inhibition of bone resorption and stimulation of renal calcium excretion, effects mediated by high affinity receptors located on osteoclasts and certain renal tubular cells. Subsequently, it was shown that CTRs are widely distributed, suggesting that CT may have more complex and diverse functions. CT may also be important in the early differentiation of the vertebrate embryo. The aims of this proposal are to complete the determination of the structure of the murine CTR gene, introduce targeted mutations into the gene by homologous recombination in embryonic stem cells and create transgenic mice that express these mutations in order to define the role of CT and the CTR gene in embryonic development. The zebrafish system will also be used to understand the role of the CTR in early embryonic development. In Specific Aim 1, determination of the structure of the murine CTR gene will be completed and the 5'-regulatory sequences identified. The murine brain CTR cDNA, sequenced in this laboratory, will be used to identify exons in the CTR gene (see Project 4). In Specific Aim 2, restriction fragments prepared from the murine CTR cDNA or the genomic CTR clones will be subcloned into vectors to produce CTR- specific cRNA probes. These will be utilized to localize CTR gene transcripts during various stages of murine development with techniques of in situ hybridization. The pattern of CTR expression determined by in situ hybridization will be correlated with that obtained by autoradiography with radioiodinated CT and immunohistochemistry with CTR antibodies. In Specific Aim 3, homologous recombination in embryonic stem (ES) will be used for targeted disruption of the CTR gene; the ES cells will be used to produce transgenic mice. This will permit direct determination of the consequences of disruption of the CTR gene in murine embryonic development. In Specific Aim 4, the lacZ gene will be placed just downstream from the translation initiation site in the CTR gene by means of homologous recombination of ES cells to produce mutant mice. This approach will permit the determination in vivo of the spatial and temporal expression of the CTR gene and the lineage of cells that express the gene during development. In Specific Aim 5, zebrafish CTR cDNA will be cloned and vectors incorporating these sequences or sequences from the CTR gene will be utilized to examine the consequences of ectopic production or overexpression of CT or the CTR in early embryonic development.

降钙素(CT)最初被认为是一种释放的多肽激素 甲状腺滤泡旁细胞对高钙血症的反应 刺激物。在哺乳动物身上，人们发现CT分泌会产生一种快速的 通过抑制骨吸收降低细胞外钙水平 和刺激肾钙排泄，高密度脂蛋白介导的效应 破骨细胞和某些肾小管上的亲和力受体 细胞。随后，研究表明，CTR分布广泛， 提示CT可能具有更复杂和多样的功能。CT可能会 在脊椎动物胚胎的早期分化中也很重要。 这项建议的目的是完成对 小鼠CTR基因的结构，将靶向突变引入 基因在胚胎干细胞中的同源重组和创造 表达这些突变的转基因小鼠，以确定其作用 CT和CTR基因在胚胎发育中的作用。斑马鱼系统 也将被用来理解CTR在早期胚胎中的作用 发展。在具体目标1中，确定 小鼠CTR基因将完成并获得5‘-调控序列 确认身份。在这个实验室测序的小鼠脑CTR基因， 将用于识别CTR基因中的外显子(见项目4)。在……里面 特异性靶向2，从小鼠CTR基因中制备限制性内切酶片段 或者将基因组CTR克隆亚克隆到载体中，以产生CTR- 特定的cRNA探针。这些将被用来定位CTR基因。 用技术在小鼠发育的不同阶段转录 原位杂交的结果。CTR表达模式由以下因素决定 原位杂交将与通过 放射性放射性CT放射自显影和CTR免疫组织化学 抗体。在特定目标3中，胚胎中的同源重组 干细胞将用于靶向干扰CTR基因； 这些细胞将被用于生产转基因小鼠。这将允许直接 CTR基因突变在小鼠体内后果的测定 胚胎发育。在特定的目标4中，LacZ基因将被放置 就在CTR基因翻译起始点的下游 ES细胞同源重组产生突变小鼠的方法。 这种方法将允许在体内确定空间和 CTR基因的时间表达和表达细胞的谱系 发育过程中的基因。在特定目标5中，斑马鱼CTR基因将 被克隆并将这些序列整合到载体中 CTR基因将被用来检测异位的后果 CT或CTR在早期胚胎中的产生或过度表达 发展。