Tet—onAdvanced调控Amelotin基因表达成釉细胞系的建立

目的应用Tet-OnAdvanced。基因表达系统构建由强力霉素（D0x）诱导Amelotin基因表达的成釉细胞系，为进一步研究Amelotin基因的生物学功能奠定基础。。方法通过fIT—PCT法从出生后7d的小鼠牙胚中克隆得到Amelotin的全长基因，测序正确后将其亚克隆入pTRE-Tight裁体中，利用Tet—onAdvanced基因表达系统先后将调控质粒pTet—OnAdvanced和反应质粒pTRE-Tight-Amelotin转入成釉细胞，分别用G418和潮霉素进行筛选．克隆扩增得到可诱导表达Amelotin基因的成釉细胞系j用RT-PCR法检测不同Dox浓度下转染成釉细胞中Amelotin基因的表达。结果连续两个回合的转染及筛选后获得了引入pTet-onAdvanced系统的细胞系，能够高诱导低背景表达Amelotin，在强力霉素诱导48h可从使Amelotin的表达显著增加，强力霉素在一定浓度范围内可以诱导Amelotin的表达呈剂量依赖性增加。结论成功构建了受强力霉素调控的双重稳定表达Amelotin．成釉细胞系，为进一步研究Amelotin与釉质矿化之间的关系奠定了基础。

Establishment of Tet-on Advanced Regulating Amelotin Expression System

Objetive To establish doxycycline（Doxl-induced Amelotin in ameloblast cell line with Tet-on Advanced regulating system in mouse ameloblast-lineage cell（ ALC ）, so as to provide an ideal experimental platform for further study of Amelotin. Methods A full length cDNA of amelotin was cloned from mouse（ postnatal 7d） tooth germ by RT-PCR and the sequence was compared with Genebank date, and then it was sub-cloned into the pTRE-Tight plasmid after sequence analysis. Tet-on Advanced regulating plasmid was transferred into ameloblast-lineage cells through G418 selection. The response plasmid of recombinant pTRE-Tight-Amelotin was transferred into positive ALC/Tet-on-Advanced cells, and stable expression of Amelotin was established through hygromycin selection. Dox was used to induce the expression of Amelotin and a cell clone sensitive to Dox was selected through RT-PCR assay. Results The expression rate of Amelotin in double stable ALC was increaseed significantly by addition of doxycyline in a concentration-dependent manner by RT-PCR,and the high expression was detected at 48h after induced by doxycyline. Conclusion Functional expression of Amelotin under Dox induced Tet regulating systerm was successfully established in ALC lines,wich provided an ideal experimental platform for further study of Amelotin.