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Generation and Initial Characterization of Osteocalcin-Deficient Rats

骨钙素缺乏大鼠的产生和初步表征

基本信息

批准号：
9146286
负责人：
Bart O Williams
金额：
$ 20.9万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-18 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9146286
关键词：
Acids Area CRISPR/Cas technology Clinic 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 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 metabolic phenotype mouse genome mouse model neurodevelopment rat genome receptor research study skeletal success therapeutic development

项目摘要

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(申请人提供)：骨钙素(OCN)是一种小蛋白，几乎只由成熟的成骨细胞和骨细胞表达，在人类中由骨γ-羧基谷氨酸包含蛋白(BGLAP)基因编码。小鼠表达骨钙素的基因由一个基因簇组成，其中该基因经历了三次复制，产生了两个骨特异的亚型(OG1和OG2)和一个在骨中不表达的亚型(Org)，这些亚型被认为是在肾脏中起作用的。此前，包括OG1和OG2的基因组缺失的纯合子小鼠被创造出来，并显示出高骨量，并出现全身代谢表型，包括葡萄糖耐量和胰岛素抵抗降低，以及男性生育能力和胎儿神经发育的变化。这项工作的一个警告是，这些小鼠产生后完成的小鼠基因组揭示了该区域内存在一个假定的基因(GM6821)，该基因编码一个与孕激素和ADIPOQ受体家族成员具有同源性的潜在剪接转录本。GM6821也通过删除小鼠OG1和OG2的基因组删除而被删除。虽然OCN缺陷小鼠使人们能够对骨钙素在小鼠生理中的作用(S)有了强有力的见解，但事实上基因组位置的组织与人类非常不同，这在将这种知识转化为临床时确实造成了潜在的并发症。虽然小鼠基因座包含复杂的三倍体，但大鼠基因组的结构类似于人类基因座，携带表达OCN/BGLAP的单个基因。因此，大鼠基因的基因组改变将更类似于人类的情况，而OCN缺陷大鼠的产生将提供一个强有力的手段来证实在小鼠模型中看到的发现，并确保OCN缺陷小鼠中伴随的GM6821缺失不会对观察到的表型做出贡献，因为大鼠和人类基因组缺乏GM6821。即使在一年前，创造OCN缺陷大鼠的提议也不可行。然而，CRISPR/CAS9系统的出现彻底改变了产生基因工程大鼠模型所需的成本和时间方面的范式。具体目的1.利用CRISPR/Cas9技术建立骨钙素缺陷型大鼠模型。我们的实验室已经使用CRISPR/Cas9来产生携带小鼠酪氨酸酶基因座高频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/BGLAP基因座来灭活该基因。特定目的2.骨钙素缺乏大鼠的骨骼和代谢表型的特征。在产生骨钙素缺乏的大鼠后，我们将确定它们是否表现出与携带大量骨钙素基因缺失的小鼠相似的骨骼和代谢表型。