CAJ | 학술논문

目的克隆人LGR5分子并构建其稳定表达的基因转染细胞株.方法提取人宫颈癌细胞株HeLa细胞总RNA,通过RT-PCR克隆获得人LGR5全长cDNA,将人LGR5全长cDNA重组入逆转录病毒载体pEGZ-term,通过293T细胞的包装获得具有感染力的完整重组病毒载体,收集培养上清感染L929细胞,筛选构建稳定表达人LGR5分子的基因转染细胞株.结果成功克隆人LGR5全长cDNA和构建pEGZ/LGR5逆转录病毒表达载体,成功获得稳定表达人LGR5的基因转染细胞株L/LGR5.结论成功克隆了人LGR5基因并构建了稳定表达LGR5分子的基因转染细胞株,为进一步研究LGR5分子的生物学功能奠定了基础.
목적극륭인LGR5분자병구건기은정표체적기인전염세포주.방법제취인궁경암세포주HeLa세포총RNA,통과RT-PCR극륭획득인LGR5전장cDNA,장인LGR5전장cDNA중조입역전록병독재체pEGZ-term,통과293T세포적포장획득구유감염력적완정중조병독재체,수집배양상청감염L929세포,사선구건은정표체인LGR5분자적기인전염세포주.결과성공극륭인LGR5전장cDNA화구건pEGZ/LGR5역전록병독표체재체,성공획득은정표체인LGR5적기인전염세포주L/LGR5.결론성공극륭료인LGR5기인병구건료은정표체LGR5분자적기인전염세포주,위진일보연구LGR5분자적생물학공능전정료기출.
Objective To clone human LGR5 full length cDNA and construct its gene transfected cell line that stably expressed the human LGR5.Methods Human LGR5 full length cDNA was cloned from human cervical cancer cell line HeLa by RT-PCR and subcloned into retroviral expressing vector pEGZ-term.The recombinant plasmid together with its helper virus vector was cotransfected into the package cell 293T with Lipofectamine 2000.The L929 cells were infected with the supernatant of the transfected 293T cells,and then were selected with Zeocin.The Zeocin resistant cells were harvested for screening their GFP and LGR5 expression by fluorescence microscope and flow-cytometry.Results Human LGR5 gene is cloned and subcloned into retroviral expressing vector pEGZ-term successfully.Gene transfected line L929/LGR5 that stably expressed the human LGR5 was constructed.Conclusion Human full length LGR5 cDNA is cloned and its retroviral expressing vector is constructed successfully.Gene transfected cell line which stably expressed the human LGR5 is established successfully.It provides a valuable tool to study the biological function of human LGR5 molecule.