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Functional analysis of AMPD gene family in cellular and animal models

AMPD基因家族在细胞和动物模型中的功能分析

基本信息

批准号：
09670171
负责人：
MORISAKI Takayuki
金额：
$ 1.98万
依托单位：
National Cardiovascular Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

AMPD gene family is thought to play an important role in purine nucleotide metabolism. To investigate functional roles of AMPD gene family in cellular and animal model, members of mouse Ampd gene family have been isolated and gene targeting experiments have been performed.First, mouse cDNA for heart-type isoform of AMPD (Ampd3) was isolated, followed by isolation of mouse cDNAs for Ampdl and Ampd3 Then, genomic DNA for corresponding Ampd gene was isolated. Sequence information revealed that mouse Ampd gene indeed functionally corresponds to a member of human AMPD gene family, respectively. Gene targeting is being performed to clarify the relationship between human AMPD1 mutation and metabolic myopathy. In addition, this expriment is expected to help to understand the recent observation that AMPD1 mutation seems to correlate better prognosis of cardiac failure.Regarding gene targeting, a targeting vector was constructed using corresponding Ampd genomic DNA as well as Neo gene and DT-A gene for positive/negative seletion. Those vectors were introduced into embryonic stem cells and G418 selected colonies were isolated. By PCR-based screening, six homologously recombined clones were establised for Ampd3 and Ampdl. Two clones of them were confirmed to carry normal karyotype. We have already obtained chimeric mice for Ampd3 or Ampdl knock-out. We are currently trying to obtain heterozygous or homozygous mice for Ampd3 or Ampdl gene knock-out. Also, we have identified new AMPD1 mutations in patients with myopaty and are currently doing functional analysis of these mutations. Further investigation of these experiments will give us a new insight of functions of Ampd gene family.

AMPD基因家族被认为在嘌呤核苷酸代谢中发挥重要作用。为了研究AMPD基因家族在细胞和动物模型中的功能作用，分离了小鼠Ampd基因家族的成员并进行了基因打靶实验。首先，分离了AMPD心脏型亚型（Ampd3）的小鼠cDNA，随后分离了Ampd1和Ampd3的小鼠cDNA，然后分离了相应Ampd基因的基因组DNA。序列信息显示，小鼠 Ampd 基因在功能上确实分别对应于人类 AMPD 基因家族的成员。正在进行基因靶向研究以阐明人类 AMPD1 突变与代谢性肌病之间的关系。此外，该实验预计将有助于理解最近观察到的AMPD1突变似乎与心力衰竭更好的预后相关。在基因打靶方面，利用相应的Ampd基因组DNA以及Neo基因和DT-A基因构建打靶载体进行正/负选择。将这些载体引入胚胎干细胞并分离G418选择的集落。通过基于PCR的筛选，为Ampd3和Ampd1建立了六个同源重组克隆。其中两个克隆被证实具有正常核型。我们已经获得了 Ampd3 或 Ampdl 敲除的嵌合小鼠。我们目前正在尝试获得用于Ampd3或Ampdl基因敲除的杂合或纯合小鼠。此外，我们还在近视患者中发现了新的 AMPD1 突变，目前正在对这些突变进行功能分析。对这些实验的进一步研究将使我们对 Ampd 基因家族的功能有新的认识。