摘要
黏蛋白1(MUC1)属黏蛋白家族成员,分布于上皮细胞膜表面,由于在免疫炎症反应以及肿瘤发生中的重要作用而日益受到重视.为了进一步深入研究MUC1的生物学功能,构建了Muc1基因敲除小鼠模型.首先,根据小鼠Muc1基因组序列设计基因剔除策略,将2个loxP位点分别插在外显子2和3两侧,构建基因剔除载体Muc1-ABRLFn-pBR322.以电穿孔方法将载体导入胚胎干细胞(ES细胞),用G418和更昔洛韦进行正负筛选获得4个同源重组的ES细胞克隆.挑选其中一个阳性ES克隆行囊胚显微注射,获得16只嵌合率大于50%的雄鼠;其次,利用嵌合雄鼠与C57BL/6J野生型雌鼠交配后获得11只floxP杂合子小鼠(10雄1雌),通过杂合子小鼠回交,并进一步与EⅡa-Cre小鼠交配,最终成功得到Muc1全身敲除小鼠,其中纯合子小鼠未出现胚胎致死现象.初步表型观察未发现Muc1基因敲除相关器官组织结构的异常改变.本研究为MUC1的生物学功能的挖掘,尤其是MUC1在肿瘤发生转移中的作用机制的揭示提供了实验平台.
MUCIN1 （MUC1）, a member of the mucin family anchored to the apical surface of the epithelial cells, is responsible for immune inflammation and tumorigenesis. A Mucl knockout mouse model was established for exploring the biological functions and studying mechanisms of MUC1 in tumorigenesis and metastasis. Mouse genomic DNA sequence of the Mucl gene was obtained using bioinformatics methods. Mucl gene knockout vector Muc1-ABRLFn- pBR322 was constructed with exon 2 and exon 3 flanked by two loxP sites. Mucl knockout vector was transferred into the embryonic stem cells by electroporation and G418 and Ganciclovoir resistant clones were screened. Four correctly homologous clones were identified by PCR, one of which was microinjected into C57BL/6J mouse blastocysts to obtain chimera mice. Sixteen mice with a chimera rate of over 50% were acquired out of 32 bred by 13 receptors. Chimeric mice were mated with C57BL/6J to obtain 11 heterozygous mice （10 males and one female）. Heterozygous mice were backcrossed and then mated with EIIa-Cre mice. Thus Mucl gene knockout mice model was successfully created and there was no embryonic lethality in homologous mutant mice. Preliminary observation of phenotype revealed no abnormalities in structures in relevant tissues and organs of Mucl knockout mice.
出处
《中国科学：生命科学》
CSCD
北大核心
2014年第2期143-150,共8页
Scientia Sinica(Vitae)
基金
国家自然科学基金(批准号:81272262)
国家科技支撑计划重点项目(批准号:2006BAI23B02)
上海市科学技术委员会项目(批准号:11140902600)资助
