摘要
目的探讨基因捕获技术应用于成体干细胞的可行性。方法线性化逆转录病毒基因捕获载体ROSAFARY,利用Lipofectamine2000转染包装细胞PA317,LacZ染色证明转染效率后,收集病毒上清,感染种植于24孔板的小鼠成肌干细胞系C2C12、人骨髓间充质干细胞(hBMSC)。为排除潮霉素B筛选的假阳性干细胞,以C2C12、hBMSC细胞基因组DNA为模板,PCR扩增ROSAFARY上LacZ部分序列(560 bp)进行鉴定。结果ROSAFARY脂质体转染PA317细胞后LacZ染色,400倍镜下可见2-5个蓝色细胞/视野。病毒上清感染种植于24孔板的C2C12和hBMSC,4 d后经LacZ染色,C2C12可见6个蓝色细胞/孔,400倍镜下hBMSC可见4-5个蓝色细胞/视野。最佳PCR鉴定参数为基因组模板量2μg,退火温度55.5℃,Mg2+浓度3 mmol/L。结论逆转录病毒基因捕获载体感染多种成体干细胞后LacZ染色显示可成功捕获激活状态启动子驱动的基因,为基因捕获应用于成体干细胞分化及恶性转化分子机制的研究奠定了基础。
Objective To investigate the possibility of gene trapping on adult stem cells. Methods Retroviral gene-trap vector, ROSAFARY, was linearized with Dra I and transfected into packaging cell line PA317 by Lipofectamine2000. The retrovirus particles containing gene-trap construct were collected to infect C2C12 and hBMSC plated in 24-well plates and LacZ staining was used to prove the efficacy of transfection. In order to exclude false drug-resistant clones of stem cells with hygromycin B selection, PCR targeting sequences （560 bp） of LacZ cDNA in ROSAFARY vector was performed using template of genomic DNA extracted from C2C12 and hBMSC. Results Under microscope with 400 magnification, 2 to 5 blue cells were found in each field by LacZ staining after transfection of PA317 cells with ROSAFARY. After C2C12 and hBMSC plated in 24-well plate were infected with retrovirus supematant for 4 d, about 6 blue cells in C2C12 per well and 4 to 5 blue cells in hBMSC in each field were observed. The optimal parameters of PCR were 2 μg genome DNA template, annealing temperature of 55.5 13, 3 mmol/L Mg^2 ＋ . Conclusion Retroviral gene-trap vector can be applied to infect many kinds of adult stem cells, and successfully trap certain activated promoters. This result provide a new platform for studying the mechanism of adult stem cells differentiation and malignant transformation by gene trap technique.
出处
《山西医科大学学报》
CAS
2008年第2期112-115,F0003,共5页
Journal of Shanxi Medical University
基金
军队"十一五"归国人员基金资助项目(06H029)
国家重点基础研究发展规划资助项目(973项目)(2005CB522605)
