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

描述（由申请人提供）：在高等脊椎动物中，髓鞘（一种包裹轴突的富含脂质的膜）极大地促进了神经传导。髓鞘由中枢神经系统中的少突胶质细胞和外周神经系统中的许旺细胞形成。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，并且高度富含两种糖脂，半乳糖神经酰胺（GalC）和硫苷脂（sGalC，GalC的3-硫酸酯）。这些髓磷脂糖脂的独特特征是它们的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-羟基在髓鞘中具有独特的作用。虽然对GalC和sGalC在髓鞘形成中的作用了解很多，但对2-羟基的具体功能还不清楚。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鞘功能中的作用。脂肪酸2-羟基化由Fa 2 h基因编码的脂肪酸2-羟基化酶催化。假设Fa 2 h基因产物负责形成2-羟基GalC/sGalC生物合成的前体，并且2-羟基脂肪酸掺入GalC/sGalC中对于髓鞘形成和髓鞘维持是关键的。目前的证据表明，Fa 2 h的表达，活性，和脂质产物（2-羟基脂肪酸）的增加，在峰值髓鞘形成期间在出生后的小鼠大脑。为了在体内测试该假设，将开发缺乏Fa 2 h基因的小鼠模型。克隆了小鼠Fa 2 h基因，并构建了靶向载体。靶向载体设计为缺失基因的外显子1，其编码Fa 2 h的脂肪酸2-羟化酶活性所需的N-末端结构域。将测定Fa 2 h敲除小鼠脑中的Fa 2 h mRNA、2-羟基脂肪酸和脂肪酸2-羟化酶活性，以确认成功的基因靶向。将构建另一种用于条件敲除的靶向载体，其将用于在ES细胞中靶向不成功、靶向ES细胞系的生殖系无能或Fa 2 h无效突变体的胚胎致死的情况。Fa 2 h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鞘功能中的作用。

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.