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，本研究首先分离了小鼠Ampd基因家族成员，并进行了基因打靶实验，首先分离了小鼠Ampd心脏型亚型（Ampd 3）的cDNA，然后分离了小鼠Ampd 1和Ampd 3的cDNA，然后分离了相应Ampd基因的基因组DNA。序列分析结果表明，小鼠AmpD基因在功能上与人类AmpD基因家族的一个成员相对应。基因靶向正在进行，以澄清人类AMPD 1突变和代谢性肌病之间的关系。在基因打靶方面，我们利用Ampd基因组DNA构建了Ampd基因打靶载体，并将Neo基因和DT-A基因进行了正反向选择。将这些载体导入胚胎干细胞，分离G418选择的集落。通过PCR筛选，获得6个Ampd 3和Ampdl同源重组克隆。其中2个克隆核型正常。我们已经获得了Ampd 3或Ampdl敲除的嵌合小鼠。我们目前正试图获得Ampd 3或Ampdl基因敲除的杂合或纯合小鼠。此外，我们已经在近视患者中发现了新的AMPD 1突变，目前正在对这些突变进行功能分析。这些实验的进一步研究将为我们对Ampd基因家族的功能提供新的认识。

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.