Creation of a fatty acid 2-hydoxylase-knockout mouse model

脂肪酸2-羟化酶敲除小鼠模型的创建

• 批准号: 7132108
• 负责人: HIROKO HAMA
• 金额: $ 7.3万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7132108
• 关键词: 3-ketosphinganine; Acyl Coenzyme A; Alleles; Anabolism; Axon; Biological; Biological Assay; Birth; Brain; Carbohydrates; Cell membrane; Ceramides; Class; Complex; Demyelinations; Dihydrosphingosine; ES Cell Line; Embryo; Environment; Esters; Exons; Fatty Acids; Figs - dietary; Future; Galactosylceramides; Gene Targeting; Genes; Genetic; Glucosylceramides; Glycolipids; Glycosphingolipids; Goals; Human; Hydrogen Bonding; Hydroxylation; Inorganic Sulfates; Knock-out; Knockout Mice; Length; Lipid Bilayers; Lipids; Maintenance; Measurement; Membrane; Messenger RNA; Methods; Mixed Function Oxygenases; Modeling; Mus; Myelin; Myelin Sheath; N-terminal; Neural Conduction; Neuraxis; Neurodegenerative Disorders; Numbers; Oligodendroglia; Palmitoyl Coenzyme A; Pathway interactions; Peripheral Nervous System; Physiological; Proteins; Rattus; Role; Sampling; Schwann Cells; Serine; Sphingolipids; Sphingomyelins; Sulfoglycosphingolipids; Testing; Thinking; Tissues; Unspecified or Sulfate Ion Sulfates; Vertebrates; aqueous; base; brain tissue; design; dihydroceramide; dihydroceramide desaturase; embryonic stem cell; fatty acid alpha-hydroxylase; hydroxy fatty acid; hydroxyl group; in vivo; mouse model; mutant; myelination; postnatal; promoter; vector

DESCRIPTION (provided by applicant): In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axons. Myelin is formed by oligodendrocytes in the central nervous system, and by Schwann cells in the peripheral nervous system. A number of devastating neurodegenerative diseases are known to cause pathological demyelination. .Myelin consists of approximately 70% lipids and 30% proteins and is highly enriched with two glycolipids, galactosylceramide (GalC) and sulfatide (sGalC, 3-sulfate ester of GalC). A unique feature of these myelin glycolipids is that approximately one half of their fatty acyl chains are 2-hydroxy fatty acids. There are no other mammalian tissues that contain such high contents of 2-hydroxy fatty acids, suggesting that the 2- hydroxyl group has a unique role in myelin. Although much is known about the role of GalC and sGalC in myelination, specific function of the 2-hydroxyl group is not well-understood. The long-term goal of this project is to determine the role of the 2-hydroxy fatty acids of GalC and sGalC in myelination and myelin function. Fatty acid 2-hydroxylation is catalyzed by fatty acid 2-hydroxylase, encoded by the Fa2h gene. It is hypothesized that the Fa2h gene product is responsible for the formation of precursors for 2-hydroxy GalC/sGalC biosynthesis, and that incorporation of 2-hydroxy fatty acids into GalC/sGalC is critical for myelination and myelin maintenance. Current evidence show that Fa2h expression, activity, and the lipid products (2-hydroxy fatty acids) increase during the peak myelination period in postnatal mouse brain. In order to test the hypothesis in vivo, a mouse model that lack Fa2h gene will be developed. The mouse Fa2h gene has been cloned, and a targeting vector has been constructed. The targeting vector is designed to delete exon 1 of the gene, which encodes the N-terminal domain required for the fatty acid 2-hydroxylase activity of Fa2h. Fa2h mRNA, 2-hydroxy fatty acids, and fatty acid 2-hydroxylase activities in the brain of Fa2h-knockout mouse will be determined to confirm successful gene targeting. Another targeting vector for conditional knockout will be constructed, which would be used in case of unsuccessful targeting in ES cells, germline-incompetence of targeted ES cell lines, or embryonic lethality of Fa2h-null mutants. The Fa2h-knockout mouse will be used in future studies to determine the role of 2- hydroxy fatty acids of GalC/sGalC in myelination and myelin function.

描述（由申请人提供）：在高等脊椎动物中，髓磷脂极大地促进了神经传导，髓磷脂是一种包裹轴突的富含脂质的膜。髓磷脂由中枢神经系统中的少突胶质细胞和周围神经系统中的雪旺细胞形成。已知许多破坏性神经退行性疾病会导致病理性脱髓鞘。髓磷脂由大约 70% 的脂质和 30% 的蛋白质组成，并且富含两种糖脂：半乳糖神经酰胺 (GalC) 和脑硫脂（sGalC，GalC 的 3-硫酸酯）。这些髓磷脂糖脂的一个独特特征是其脂肪酰基链的大约一半是 2-羟基脂肪酸。没有其他哺乳动物组织含有如此高含量的 2-羟基脂肪酸，这表明 2-羟基在髓磷脂中具有独特的作用。尽管人们对 GalC 和 sGalC 在髓鞘形成中的作用了解很多，但 2-羟基的具体功能尚不清楚。该项目的长期目标是确定 GalC 和 sGalC 的 2-羟基脂肪酸在髓鞘形成和髓磷脂功能中的作用。脂肪酸 2-羟基化由 Fa2h 基因编码的脂肪酸 2-羟化酶催化。据推测，Fa2h 基因产物负责 2-羟基 GalC/sGalC 生物合成前体的形成，并且 2-羟基脂肪酸掺入 GalC/sGalC 对于髓鞘形成和髓磷脂维持至关重要。目前的证据表明，Fa2h 表达、活性和脂质产物（2-羟基脂肪酸）在出生后小鼠大脑的髓鞘形成高峰期增加。为了在体内验证这一假设，我们将开发一种缺乏 Fa2h 基因的小鼠模型。小鼠Fa2h基因已克隆，并构建了靶向载体。靶向载体旨在删除该基因的外显子 1，该外显子编码 Fa2h 脂肪酸 2-羟化酶活性所需的 N 末端结构域。将测定 Fa2h 敲除小鼠大脑中的 Fa2h mRNA、2-羟基脂肪酸和脂肪酸 2-羟化酶活性，以确认基因靶向成功。将构建另一种用于条件敲除的靶向载体，该载体将用于 ES 细胞中靶向失败、靶向 ES 细胞系的种系无能或 Fa2h 缺失突变体的胚胎致死性的情况。 Fa2h 敲除小鼠将用于未来的研究，以确定 GalC/sGalC 的 2-羟基脂肪酸在髓鞘形成和髓磷脂功能中的作用。