Functional analysis of AMPD gene family in cellular and animal models

AMPD基因家族被认为在嘌呤核苷酸代谢中起重要作用。为了研究AMPD基因家族在细胞和动物模型中的功能作用，我们分离了小鼠AmPD基因家族的成员，并进行了基因打靶实验。首先，分离了小鼠心型AMPD基因亚型(Ampd3)的cDNA，然后分离了小鼠Ampd1和Ampd3的cDNA，然后提取了相应的Ampd基因的基因组DNA。序列信息显示，小鼠AmpD基因在功能上确实与人类AMPD基因家族中的一个成员相对应。基因打靶正在被用来阐明人类AMPD1突变和代谢性肌病之间的关系。在基因打靶方面，我们利用相应的AmpD基因组DNA以及Neo基因和DT-A基因构建了基因打靶载体，进行阳性/阴性选择。将这些载体导入胚胎干细胞，分离出G418克隆。通过聚合酶链式反应筛选，获得了6个Ampd3和Ampd1的同源重组克隆。其中两个克隆被证实具有正常的核型。我们已经获得了用于Ampd3或Ampd1基因敲除的嵌合小鼠。我们目前正试图获得Ampd3或Ampd1基因敲除的杂合子或纯合子小鼠。此外，我们已经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基因家族的功能有一个新的认识。

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.