CALCITONIN RECEPTORS IN DEVELOPMENT
CALCITONIN RECEPTORS IN DEVELOPMENT
批准号:
6100314
负责人:
STEVEN GOLDRING
金额:
$0.0万
依托单位国家:
美国
项目类别:
财政年份:
1997
资助国家:
美国
项目状态:
已结题
起止时间:
1997-01-01 至 1998-12-31
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
中文摘要
降钙素 (CT) 最初被鉴定为释放的肽激素
甲状腺滤泡旁细胞响应高钙血症
刺激。 在哺乳动物中,发现 CT 分泌会产生快速的
通过抑制骨吸收降低细胞外钙水平
和刺激肾钙排泄,高介导的影响
亲和受体位于破骨细胞和某些肾小管上
细胞。 随后,结果表明点击率分布广泛,
表明CT可能具有更加复杂和多样化的功能。 CT检查可能
在脊椎动物胚胎的早期分化中也很重要。
本提案的目的是完成
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基因并创建
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
还将用于了解 CTR 在早期胚胎中的作用
发展。 在具体目标 1 中,确定
小鼠CTR基因将完成并且5'-调控序列
确定。 小鼠大脑 CTR cDNA,在本实验室测序,
将用于识别 CTR 基因中的外显子(参见项目 4)。 在
具体目标2,从鼠CTR cDNA制备限制性片段
或者将基因组 CTR 克隆亚克隆到载体中以产生 CTR-
特异性cRNA探针。 这些将用于定位 CTR 基因
使用技术记录小鼠发育各个阶段的转录本
的原位杂交。 CTR表达模式由下式确定
原位杂交将与获得的结果相关联
放射性碘 CT 放射自显影和 CTR 免疫组织化学
抗体。 在具体目标 3 中,胚胎中的同源重组
干(ES)将用于有针对性地破坏CTR基因; ES
细胞将用于生产转基因小鼠。 这将允许直接
确定小鼠 CTR 基因破坏的后果
胚胎发育。 在特定目标 4 中,lacZ 基因将被放置
就在 CTR 基因翻译起始位点的下游
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测定空间和
CTR 基因的时间表达和表达细胞的谱系
发育过程中的基因。 在具体目标 5 中,斑马鱼 CTR cDNA 将
被克隆并包含这些序列或来自以下序列的载体
CTR基因将用于检查异位的后果
CT 或 CTR 在早期胚胎中的产生或过度表达
发展。
英文摘要
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.
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