DEVELOPING ATHEROGENESIS BY GENE TARGETING

通过基因靶向发展动脉粥样硬化

基本信息

批准号：
2228443
负责人：
Jorge A Piedrahita
金额：
$ 20.46万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-12-15 至 1997-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2228443
关键词：
apolipoproteins arteriosclerosis cell bank /registry cell differentiation disease /disorder model embryo /fetus embryo /fetus tissue /cell culture embryonic stem cell familial hyperlipoproteinemia type III genetic library genetic models model design /development molecular cloning nucleic acid sequence recombinant DNA restriction fragment length polymorphism swine

项目摘要

In the United States cardiovascular disease is the major cause of disabilities and accounts for approximately half of all deaths each year. The disease is complex with both genetic and epigenetic factors influencing its severity. Moreover, the genetic factors are not well understood due to the number of genes involved and the difficulty in accounting for the multiple environmental influences effecting the rate of cardiovascular disease on the normal population. Because of its complexity it would be of great benefit to have animal systems, were the environmental effects can be carefully controlled, with known modification in genes thought to be involved in the development of atherogenesis. Recently the ability to modify specific genes in mouse embryonic stem cells by homologous recombination, combined with the ability of the modified ES cells to contribute to the formation of the germ cells in ES- Blastocyst chimeras, has opened a new venue with which to generate animal models of cardiovascular disease. This technology has been utilized by us to generate a mouse model of familial typeIII hyperlipo-proteinemia and premature atherosclerosis by inactivation of the apolipoprotein E gene. It would be of great interest to generate a porcine model with apoE deficiency to complement the existing mouse model. Unavailability of stable ES cell lines from forcing origin prevents us from doing so, however. The overall objective of this application include: 1) the isolation and characterization of stable cell line of porcine embryonic stem cells. Porcine ES cells will be isolated and maintained in an undifferentiated state by addition of recombinant porcine leukemia inhibitory factor (pLIF) to the culture media. Isolated cell lines will be characterized for their ability to differentiate in vitro and for their ability to generate germ line chimeras; 2) the cloning and molecular characterization of the porcine apolipoprotein-E gene. The apoE gene will be isolated from porcine genomic library and sequenced to determined overall homology to the human and the mouse gene. Additionally, DNA polymorphisms at the apoE locus will be determined by single stranded conformational polymorphisms and heteroduplex DNA analysis; 3) development of a porcine model of familial type III hyperlipoproteinemia. The inactivation of the porcine apoE gene will be accomplished by a homologous recombination event resulting in insertion of the neo gene in exon 2. The resulting animal model will not only complement the existing mouse model of apoE deficiency but will also provide the opportunity for looking at phenotypic changes due to apoE deficiency that are either not evident in mice or difficult to study in a small animal model such as the mouse.

在美国，心血管疾病是每年所有死亡的残疾和占一半。该疾病与遗传和表观遗传因子很复杂影响其严重性。而且，遗传因素不好由于涉及的基因数量和困难而理解计算影响率的多种环境影响正常人群的心血管疾病。因为它复杂性，拥有动物系统将是很大的好处可以仔细控制环境效果，并已知被认为与发展有关的基因的修改动脉粥样硬化。最近修改小鼠胚胎茎中特定基因的能力通过同源重组的细胞，结合了修饰ES细胞有助于在ES-中形成生殖细胞胚泡嵌合体开设了一个新的场所，以产生动物心血管疾病的模型。这项技术已被我们生成一个家族性型Hyperlipo-蛋白血症的鼠标模型和过早的动脉粥样硬化，通过失活基因。与 APOE缺乏以补充现有鼠标模型。不可用稳定的ES细胞线的强迫来源阻止了我们这样做，然而。本应用程序的总体目标包括：1）隔离和猪胚胎干细胞稳定细胞系的表征。猪ES细胞将被分离并维持在未分化的通过添加重组猪白血病抑制因子的状态（PLIF）到培养基。孤立的细胞系将被表征因为他们有能力在体外和能力产生种系嵌合体； 2）克隆和分子猪载脂蛋白-e基因的表征。 apoe基因将从猪基因组文库中分离出来，并确定确定与人和小鼠基因的总体同源。另外，DNA APOE基因座的多态性将由单链确定构象多态性和杂化DNA分析； 3）家族性高脂蛋白血症家族性猪模型的发展。猪APOE基因的失活将通过A实现同源重组事件导致插入NEO基因外显子2。由此产生的动物模型不仅会补充现有的 APOE缺乏症的鼠标模型，但也将为查看由于APOE缺乏症而不是不是的表型变化在小鼠中明显或在小动物模型中很难研究老鼠。