Generation and Initial Characterization of Osteocalcin-Deficient Rats

骨钙素缺乏大鼠的产生和初步表征

• 批准号: 9042638
• 负责人: Bart O Williams
• 金额: $ 25.08万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9042638
• 关键词: 1-Carboxyglutamic Acid; Area; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Sequence Alteration; Databases; Diabetes Mellitus; Double Strand Break Repair; Ensure; Enzymes; Family; Fertility; Frequencies; Gene Cluster; Gene Proteins; Generations; Genes; Genetic Engineering; Genome; Genomics; Goals; Health; Hormones; Human; Human Genome; Insulin Resistance; Kidney; Knowledge; Laboratories; Metabolic; Methods; Michigan; Microinjections; Modeling; Monophenol Monooxygenase; Mus; Nonhomologous DNA End Joining; Osteoblasts; Osteocalcin; Osteocytes; Osteoporosis; Phenotype; Physiological; Physiology; Progestins; Protein Isoforms; Proteins; RNA Splicing; Rattus; Research; Role; Skeletal Development; Structure; System; Technology; Time; Transcript; Transgenic Organisms; Translating; Universities; Work; base; blood glucose regulation; bone; bone mass; cost; experience; fetal; glucose metabolism; glucose tolerance; insertion/deletion mutation; insight; male; member; mouse genome; mouse model; neurodevelopment; public health relevance; rat genome; receptor; research study; skeletal; success; therapeutic development

 DESCRIPTION (provided by applicant): Osteocalcin (Ocn), encoded in humans by the bone gamma-carboxyglutamic acid-containing protein (BGLAP) gene, is a small protein almost exclusively expressed by mature osteoblasts and osteocytes. The mouse locus expressing osteocalcin consists of a gene cluster where the gene has undergone a triplication resulting in two bone-specific isoforms (Og1 and Og2) and one isoform (Org) not expressed in bone proposed to function in the kidney. Previously, mice homozygous for a genomic deletion encompassing both Og1 and Og2 were created and displayed high bone mass and developed systemic metabolic phenotypes including decreased glucose tolerance and insulin resistance as well as alterations in male fertility and fetal neural development. A caveat to this work is that te completion of the mouse genome subsequent to the generation of these mice revealed the presence of a putative gene within this region (GM6821) that encodes a potentially spliced transcript with homology to members of the progestin and adipoQ receptor family. GM6821 is also removed via the genomic deletion that removes mouse Og1 and Og2. While the Ocn-deficient mice have allowed powerful insights into the role(s) of osteocalcin in mouse physiology, the fact the genomic locus is organized very differently from the human does create potential complications in translating this knowledge to the clinic. While the mouse locus contains a complex triplication, the rat genome is structured similarly to the human locus, carrying a single gene expressing OCN/BGLAP. Thus, genomic alteration of the rat gene would be more analogous to the human situation and the generation of Ocn-deficient rats would provide a powerful means to confirm the findings seen in the mouse models and ensure that the concomitant deletion of GM6821 in the Ocn-deficient mouse is not contributing to the observed phenotypes since the rat and human genomes lack GM6821. A proposal to create Ocn-deficient rats would not have been feasible even a year ago. However, the availability of the CRISPR/Cas9 system completely changes the paradigm in terms of the cost and time required to generate genetically engineered rat models. Specific Aim 1. Use CRISPR/Cas9 technology to create osteocalcin-deficient rats. Our laboratory has used CRISPR/Cas9 to generate mice carrying high frequency biallelic deletions in the murine tyrosinase locus. We will carry out an analogous experiment to target the rat Ocn/BGLAP locus to inactivate the gene. Specific Aim 2. Characterize skeletal and metabolic phenotypes in osteocalcin-deficient rats. After generating Osteocalcin-deficient rats, we will determine if they display skeletal and metabolic phenotypes similar to those observed in mice carrying the large deletion of the osteocalcin locus.

 描述（由申请人提供）：骨钙素（Ocn）在人体中由骨γ-羧基谷氨酸蛋白（BGln）基因编码，是一种几乎仅由成熟成骨细胞和骨细胞表达的小蛋白。表达骨钙素的小鼠基因座由一个基因簇组成，其中该基因经历了三重化，产生两种骨特异性亚型（Og 1和Og 2）和一种在骨中不表达的亚型（Og 1），建议在肾脏中发挥作用。以前，创建了包含Og 1和Og 2的基因组缺失的纯合子小鼠，并显示出高骨量，并形成了全身代谢表型，包括葡萄糖耐量降低和胰岛素抵抗以及雄性生育力和胎儿神经发育的改变。这项工作的一个警告是，在这些小鼠产生之后完成的小鼠基因组揭示了在该区域内存在一个推定的基因（GM 6821），该基因编码一个潜在的剪接转录物，该转录物与阿替米星蛋白和adipoQ受体家族的成员具有同源性。GM 6821也通过去除小鼠Og 1和Og 2的基因组缺失而被去除。虽然Ocn缺陷小鼠已经允许对骨钙素在小鼠生理学中的作用进行强有力的洞察，但基因组位点的组织与人类非常不同的事实确实在将这些知识转化为临床时产生了潜在的并发症。虽然小鼠基因座含有复杂的三重序列，但大鼠基因组的结构与人类基因座相似，携带表达OCN/BGly的单个基因。因此，大鼠基因的基因组改变将更类似于人类情况，并且Ocn缺陷大鼠的产生将提供有力的手段来确认在小鼠模型中观察到的发现，并确保在Ocn缺陷小鼠中伴随的GM 6821缺失不会对观察到的表型有贡献，因为大鼠和人类基因组缺乏GM 6821。一年前，创造OCN缺陷大鼠的提议是不可行的。然而，CRISPR/Cas9系统的可用性完全改变了生成基因工程大鼠模型所需的成本和时间方面的范式。具体目标1。使用CRISPR/Cas9技术创建骨钙素缺陷大鼠。我们的实验室已经使用CRISPR/Cas9来产生在鼠酪氨酸酶基因座中携带高频率双等位基因缺失的小鼠。我们将进行一个类似的实验，以靶向大鼠Ocn/BGln基因座来定位该基因。具体目标2。骨钙素缺乏大鼠的骨骼和代谢表型特征。在产生骨钙素缺陷大鼠后，我们将确定它们是否显示出与携带骨钙素基因座大缺失的小鼠中观察到的相似的骨骼和代谢表型。