Transgenic mouse as a model of lipoprotein glomerulopathy (LPG), and analysis of LPG

脂蛋白肾病(LPG)是1988年新发现的一种肾脏疾病。其特点是肾小球毛细血管明显扩张，脂蛋白沉积异常，血浆载脂蛋白E浓度升高。最近，我们在三个日本家系中发现了载脂蛋白E的变异体--仙台(Arg 145 Pro)。此外，我们还发现了三个不同的新的载脂蛋白E变异体，即载脂蛋白E2京都(Arg25Cys)、载脂蛋白E1(del 141-143)和载脂蛋白E1(del 156-173)。建立载脂蛋白E1(del 141-143)转基因小鼠作为LPG的动物模型，利用定点突变技术将apoE1(del 141-143)突变引入正常apoE基因启动子区域(11.5 kb)。用HindIII酶切人载脂蛋白E基因，纯化后显微注射到C57BL/6小鼠受精卵的原核中。注射的卵子被手术转移到麻醉的代孕雌性的输卵管中。然而，转基因小鼠因感染了致病病毒而被杀死。进行第二次微量注射，产下7只幼崽。从它们的尾巴中提取基因组DNA。用人载脂蛋白E基因的引物对基因组尾部DNA进行聚合酶链式反应分析。人载脂蛋白E基因是从一只雌性小鼠身上检测到的。人载脂蛋白E基因在F1小鼠中的检出率很低，目前在两只雄性小鼠中证实了人载脂蛋白E基因。在建立apoE1(del 141-143)小鼠的纯合子后，计划分析它们的肾小球病变和血清脂蛋白。

Lipoprotein glomerulopathy (LPG) is a newly recognized renal disease in 1988. It is characterized by abnormal lipoprotein deposition in the marked dilated glomerular capillaries, and elevated plasma concentration of apolipoprotein (apo) E.Recently, we identified an apoE variant, apoE2 Sendai (Arg 145Pro) in three Japanese kindred with LPG.Furthermore, we discovered three different novel apoE variants, apo E2 Kyoto (Arg25Cys) apo E1 (del 141-143) and apo E1 (del 156-173), which are closely related to LPG.Establishment of an apo E 1 (del 141-143) transgenic mouse was tried as an animal model of LPG.ApoE1 (del 141-143) variant was introduced into normal apoE gene including promoter region (11.5kb) using site directed mutagenesis. Fragment of the variant human apoE gene digested with HindIII was purified and microinjected into pronuclei of fertilized eggs of C57BL/6 mice. Injected eggs were surgically transferred to oviducts of anesthetized surrogate females. However, the transgenic mice were killed because they were infected with pathogenic virus. Second microinjection was done, and seven pups were born. Genomic DNA was isolated from their tails. Polymerase chain reaction analysis was performed on genomic tail DNA using primer set of human apoE gene. Human apoE gene was detected from a female mouse. Human apoE gene detection rate of Fl mice was very low, and human apoE gene was confirmed currently in two male mice. After homozygotes of apoE1 (del 141-143) mice are established, analysis of their glomerulus lesions and serum lipoproteins are planned.