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，髓磷脂极大地促进了神经传导，髓磷脂是包裹在轴突周围的一种富含脂质的膜。髓磷脂由中枢神经系统的少突胶质细胞和周围神经系统的雪旺细胞形成。已知许多毁灭性的神经退行性疾病可引起病理性脱髓鞘。髓磷脂由大约70%的脂质和30%的蛋白质组成，并富含两种糖脂，半乳糖神经酰胺（GalC）和硫脂（sGalC， GalC的3-硫酸盐酯）。这些髓磷脂糖脂的一个独特特征是，大约一半的脂肪酰基链是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甚多，但2-羟基的具体功能尚不清楚。该项目的长期目标是确定2-羟基脂肪酸GalC和sGalC在髓鞘形成和髓鞘功能中的作用。脂肪酸2-羟基化是由脂肪酸2-羟基化酶催化的，由Fa2h基因编码。假设Fa2h基因产物负责2-羟基GalC/sGalC生物合成前体的形成，并且2-羟基脂肪酸与GalC/sGalC的结合对于髓鞘形成和髓磷脂维持至关重要。目前的证据表明，Fa2h的表达、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，将开发缺乏Fa2h基因的小鼠模型。克隆了小鼠Fa2h基因，构建了靶向载体。该靶向载体被设计用于删除该基因的外显子1，该基因编码Fa2h脂肪酸2-羟化酶活性所需的n端结构域。Fa2h敲除小鼠脑内Fa2h mRNA、2-羟基脂肪酸和脂肪酸2-羟化酶活性的测定将证实基因靶向成功。将构建另一个条件敲除的靶向载体，用于胚胎干细胞靶向失败、靶向胚胎干细胞系不正常或Fa2h-null突变体的胚胎致死性。fa2h敲除小鼠将在未来的研究中用于确定2-羟基脂肪酸GalC/sGalC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.