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Molecular basis and approach to novel therapeutic strategies for mucopolysaccharidosis IVA - genomic cloning of mouse Galns and development of mouse model for MPSIVA -

粘多糖贮积症 IVA 新治疗策略的分子基础和方法 - 小鼠 Galns 的基因组克隆和 MPSIVA 小鼠模型的开发 -

基本信息

批准号：
09670858
负责人：
ORII Tadao
金额：
$ 2.37万
依托单位：
Gifu University
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 1999
项目状态：
已结题

项目摘要

In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kbcDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84%similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GA/AC consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90bp from the ATG codon. The 5'-flanking region lackes canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.We have constructed a plasmid containing the Galns gene fragment in the Neo+TK vector. The targeting vector introduced into the ES cells. Targeted clones were isolated. Targeted ES cells lines were injected into the blastcytes of embryos. At now, highly chimeric male mice are going to obtain.

为了促进使用 MPS IV A 模型动物进行体内研究，我们分离并进行了人 GALNS 小鼠同源物的分子表征。 2.3-kbcDNA 包含编码 520 个氨基酸残基的 1560-bp 开放阅读框。该编码区在氨基酸水平上与人GALNS cDNA有84%的相似性。通过种间回交分析将小鼠 Galns 基因定位到 8 号染色体的远端区域，在此与 Aprt 共分离。 Northern印迹分析显示单拷贝基因的广泛表达，尤其是在肝脏和肾脏中表达较高。 Galns 基因长约 50 kb，由 14 个外显子和 13 个内含子组成。除外显子 8/内含子 8 连接外，所有内含子-外显子剪接连接均符合 GA/AC 共有序列。引物延伸显示-44和-75之间有多个转录起始位点，尽管在距ATG密码子-90bp处观察到主要转录起始位点。 5'-侧翼区域缺乏典型的 TATA 和 CAAT 盒序列，但富含 G+C，含有 10 个 GC 盒（潜在的 Sp1 结合位点），这是管家基因启动子的特征。我们在 Neo+TK 载体中构建了包含 Galns 基因片段的质粒。将靶向载体引入 ES 细胞。分离目标克隆。将靶向 ES 细胞系注射到胚胎的胚泡中。目前，高度嵌合的雄性小鼠即将获得。