人乳腺癌LINE-1稳转细胞系的建立

Construction of LINE-1 Overexpression Stable Cell Line of Human Breast Cancer

目的构建人乳腺癌细胞MDA-MB-231稳定过表达LINE-1（long interspersed nuclear element1）细胞系，为研究LINE-1在乳腺癌的发生发展中的作用提供新的细胞模型。方法将带有neo（neomycin）抗性基因的LINE-1表达质粒瞬时转染到细胞中，当质粒稳定整合到基因组时细胞能表达出Neo基因并获得抗性产物，这类细胞则能在含有G418的选择性培养基中存活下来；存活下来的细胞进行有限稀释得到单个细胞，条件培养基培养单细胞至长成单细胞克隆；采用普通PCR、琼脂糖凝胶电泳、Real time PCR对得到的单细胞克隆进行稳转效果验证。结果通过质粒转染、G418药物筛选和有限稀释法得到LINE-1稳定高表达乳腺癌细胞系。结论成功建立了人乳腺癌细胞LINE-1稳定高表达细胞系，为后续研究提供了细胞模型。

Objective To establish stable Long Interspersed Nuclear Element 1 （LINE-1） - overexpression cell line in human breast cancer cell MDA-MB-231 for the investigation of LINE-1 in breast cancer development and progression. Methods LINE-1 expression plasmid with neomycin resistance gene was transfected into cells followied by G418 selection. The stable LINE-1-Neo expression cells survived. Then limiting dilution assay was used to obtain a single cell which was then incubated with conditioned medium until it grew into clone. The stable transfection efficiency was then verified using PCR and Real time PCR. Results LINE-1 stable over-expression cell lines were obtained by plasmid transfection and G418 drug screening and limiting dilution. Conclusion The LINE-1 stable over-expression cell lines were successfully established which provided a new cell model for further study of the LINE-1 function in breast cancer.