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

为了便于使用模型动物进行MPS IV A的体内研究，我们分离了人类GALNS的小鼠同源物并进行了分子表征。2.3 kbcdna包含一个1560 bp的开放阅读框，编码520个氨基酸残基。该编码区与人类GALNS cDNA在氨基酸水平上的相似性为84%。通过种间回交分析，将小鼠Galns基因定位到与Aprt共分离的8号染色体远端区域。Northern blot分析显示一个单拷贝基因广泛表达，尤其是在肝脏和肾脏中表达量更高。Galns基因长约50kb，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-外显子剪接均符合GA/AC一致序列。引物延伸显示多个转录起始位点在-44 ~ -75之间，但主要转录起始位点在ATG密码子-90bp处。5'-侧翼区域缺乏典型的TATA和CAAT盒序列，但富含G+C，具有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载体上构建了含有Galns基因片段的质粒。将靶向载体导入胚胎干细胞。分离出目标克隆。将目标胚胎干细胞注入胚胎胚细胞。目前，高度嵌合的雄性小鼠将获得。

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.