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

为了便于利用模型动物进行MPS IV A的体内研究，我们分离并进行了人GALN的小鼠同源基因的分子鉴定。2.3kbcDNA包含一个1560bp的开放阅读框，编码520个氨基酸残基。该编码区与人GALNS基因在氨基酸水平上有84%的同源性。通过种间回交分析，将小鼠GALNS基因定位于8号染色体的远端区域，在那里它与aprt共分离。Northern印迹分析表明，单拷贝基因广泛表达，尤其是在肝脏和肾脏中表达水平较高。该基因全长约50kb，由14个外显子和13个内含子组成。除外显子8/内含子8外，其余内含子-外显子剪接均符合GA/AC一致序列。引物延伸显示-44~-75之间有多个转录起始点，但主要的转录起始点在ATG密码子的-90bp处。5‘-侧翼区缺乏典型的TATA和CAAT box序列，但G+C富含10个GC box(潜在的Sp1结合位点)，具有看家基因启动子的特征。将靶向载体导入ES细胞。分离出目标克隆。将靶向ES细胞系注射到胚胎的囊胚细胞中。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.