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）最初被鉴定为释放的肽激素 通过甲状腺的副细胞响应高钙血症 刺激。 在哺乳动物中，发现CT分泌产生了快速 通过抑制骨吸收来降低细胞外钙水平 和肾脏钙排泄的刺激，由高介导的作用 位于破骨细胞和某些肾小管上的亲和力受体 细胞。 随后，表明CTR分布广泛， 表明CT可能具有更复杂和多样化的功能。 CT可能 在脊椎动物胚胎的早期分化中也很重要。 该提案的目的是完成确定 鼠CTR基因的结构将靶向突变引入 通过在胚胎干细胞中同源重组的基因，并创建 表达这些突变以定义角色的转基因小鼠 CT和CTR基因的胚胎发育。 斑马鱼系统 还将用于了解CTR在早期胚胎中的作用 发展。 在特定目标1中，确定结构 鼠CTR基因将完成，并进行5'调节序列 确定。 在该实验室中测序的鼠脑CTR cDNA， 将用于识别CTR基因中的外显子（请参阅项目4）。 在 特定的目标2，由鼠CTR cDNA制备的限制片段 否则基因组CTR克隆将被亚克隆到载体中，以产生ctr- 特定的CRNA探针。 这些将用于定位CTR基因 在鼠开发的各个阶段的成绩单 原位杂交。 CTR表达的模式由 原位杂交将与 带有放射性约束的CT和免疫组织化学的放射自显影 抗体。 在特定的目标3中，胚胎中的同源重组 茎（ES）将用于靶向破坏CTR基因； ES 细胞将用于产生转基因小鼠。 这将允许直接 确定鼠中CTR基因破坏的后果 胚胎发展。 在特定的目标4中，将放置LACZ基因 只是从CTR基因的翻译起始位点下游 ES细胞同源重组以产生突变小鼠的手段。 这种方法将允许在空间的体内确定 CTR基因的时间表达和表达细胞的谱系 发育过程中的基因。 在特定的目标5中，斑马鱼CTR cDNA将 克隆并从 CTR基因将用于检查异位的后果 早期胚胎中CT或CTR的生产或过表达 发展。