小鼠GM─CSFcDNA重组逆转录病毒的构建及在成纤维细胞中的表达

为探索粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因治疗的可能性，将小鼠ＧＭ－ＣＳＦｃＤＮＡ重组于缺陷型逆转录病毒载体ｐＬＸＳＮ中，重组质粒转染病毒包装细胞ＰＡ３１７，经Ｇ４１８筛选，用其抗性克隆培养上清液感染小鼠成纤维细胞ＮＩＨ３Ｔ３，筛选出抗Ｇ４１８的转化细胞克隆，用ＰＣＲ和Ｓｏｕｔｈｅｒｎｂｌｏｔ证明转化细胞的基因组中整合有ＮｅｏＲ基因和ＧＭ－ＣＳＦ基因，原位杂交显示转化细胞有较强的ＧＭ－ＣＳＦｍＲＮＡ表达，用骨髓细胞增殖法和ＣＦＵ－ＧＭ检测也表明转化细胞分泌有较多的ＧＭ－ＣＳＦ，该研究为下一步在动物体内进行ＧＭ－ＣＳＦ基因治疗的研究奠定基础

Construction of granulocyte/macrophage colony stimulating factor cDNA recombinant retrovirus and its expression in fibroblasts of mice

In order to explore the feasibility of granulocyte/macrophage colony stimulating factor (GM CSF), murine GM CSF cDNA was recombined with retrovirus vector pLXSN and then the recombinant plasmid was transfected into retrovirus packaging cell line PA317. After G418 screening, the supernatant of cultured G418 resistant clones was used to infect murine fibroblast NIH3T3 and the G418 resistant transforming cell clone was obtained. PCR and Southern blot hybridization demonstrated integrated NeoR and GM CSF genes in the genome of the transforming cells and strong expression of GM CSF mRNA was found by in situ hybridization. The technique of marrow cells proliferation and CFU GM detection also confirmed that the transforming cells secreted a great deal of GM CSF. Our study provides a basis for the further study of gene therapy of GM CSF in animals.