快速分离和鉴别转基因单克隆细胞的改良方法  

Improved methods to fast separation and identification of genetically modified monoclonal cells

作　　者：王亚婷[1] 李新娜[2] 张丽娜[2] 郭芳 杜鹃[4] 赵溶冰 张正银[1] 周晓娜[2] 汪晶冰[2] 李剑华[2] 刘敏[2] 方宏罡[2] 

机构地区：[1]上海市浦东新区浦南医院检验科分子诊断室,上海200125 [2]黑龙江省大庆市大庆油田总医院检验科细胞遗传室,大庆163001 [3]浦东新区金杨社区卫生服务中心口腔科,上海200135 [4]黑龙江省大庆市大庆油田总医院肾内科,大庆163001 [5]黑龙江省大庆油田脑血管医院脑内科,大庆163113

出　　处：《中国优生与遗传杂志》2016年第4期36-39,F0003,共5页Chinese Journal of Birth Health & Heredity

基　　金：上海市浦东新区卫生系统优秀学科带头人培养计划(PWRd2015-08);黑龙江省青年科学基金项目(QC2013C119)

摘　　要：目的探索快速有效的从稳定转染后的悬浮多克隆细胞中分离、纯化并鉴定出单克隆的技术改良法。方法电穿孔法稳定转染后的T淋巴细胞系先在分离培养基中扩增成多克隆,用有限稀释法分离出单个细胞,一部分用丝裂霉素C处理的饲养细胞培养扩增单克隆,一部分进行无饲养细胞扩增,用未转染的T淋巴细胞系的新鲜培养上清换液,对比两法的单克隆得率。检测单克隆的靶基因整合则先用荧光素酶报告实验再结合引物PCR扩增法,筛选出表达标记蛋白并整合有完整特异基因片段的单克隆。结果两种方法在分离培养基中存活增殖的活细胞克隆数量比例分别为5.27%和4.55%,无显著差异,通过荧光素酶报告基因实验确定完整整合外源基因的靶克隆比率(0.80%和0.57%)也无显著差异。单克隆的报告荧光素酶表达强度显著高于多克隆和对照,比较直观快速。结论无饲养细胞法避免了饲养细胞复燃污染单克隆细胞,简化了步骤提高了效率,能保证单个细胞在选择培养基中成功扩增出单克隆。荧光素酶报告基因实验能快速、有效检测出外源整合基因的表达。Objective：Effective methods and technology were explored for getting simple and fast separation,purification and identify of monoclonal cells from stable transfected suspended polyclonal cells. Methods：Single cells were cultured in selective medium by limited dilution from polyclones after electroporation of T cell lines. The separation rates of amplified monoclonal cells were compared between feeder cells method and no feeder cells isolation method. luciferase report assay was employed to determine the fluorescence marker protein of the candidate monoclones. Monoclons with expression of selected marker protein and integration of complete specific gene fragments were screened and identified. Results：There was no significant difference of survival monocells including fake monoclonal cells between feeder and no feeder isolation methods. The real target monoclones with integration of complete exogenous genes were further determined by luciferase report assay in those clones. Also no significant difference was found in the isolation rates. Fluorescent intensity of monoclons is significantly higher than polyclonal cells expression. Marker protein is effective and fast signals to distinguish the target clones. Conclusion：Pollution of feeders is avoided in no feeder isolation method. Its less time consuming,simplified steps and improved efficiency. Luciferase report assay can quickly and available to detect the expression of integrity and specific of exogenous genes in genetically modified monoclonal cells.

关 键 词：饲养细胞 单克隆 培养上清 荧光素酶报告实验 丝裂霉素C 

分 类 号：R394[医药卫生—医学遗传学]