细胞转染中单克隆与混克隆干扰基因效率的比较  

Comparative study on the efficiency of gene interference between monoclonal and mixed clone cell during cell transfection

作　　者：刘燕[1] 朱吉海[2] 孙伟[1] 赵珺[1] LIU Yan ZHU Ji-hai SUN Wei ZHAO Jun(Department of Immunology, Qinghai University Medical College Department of Cardiothrocic Surgery, the Affiliated hospital of Qinghai University)

机构地区：[1]青海大学医学院免疫学教研室 [2]青海大学附属医院心胸外科

出　　处：《青海医学院学报》2016年第4期263-267,共5页Journal of Qinghai Medical College

基　　金：国家自然科学基金(编号:81550043);青海省自然科学基金青年项目(编号:2015-ZJ-940Q)

摘　　要：目的比较细胞转染中单克隆与混克隆干扰基因效率。方法采用胃泌素特异干扰载体转染人胃癌原代细胞株L25,经G418抗性筛选获得混克隆和单克隆细胞,应用RT-PCR、Western blot和免疫荧光三种方法对混克隆细胞和单克隆细胞干扰胃泌素基因的效率进行比较。结果 RT-PCR结果显示混克隆细胞胃泌素mRNA表达水平被明显抑制。在挑取的10株单克隆细胞中,胃泌素的表达均受到干扰,其效果有差异,其中有2株单克隆细胞pshRNA-1、pshRNA-10胃泌素mRNA表达水平抑制率达到86%,抑制效率与混克隆细胞相同,其他8株单克隆细胞的平均抑制率为31%。Western blot和免疫荧光结果均显示单克隆细胞pshRNA-1、pshRNA-10与混克隆细胞在胃泌素蛋白水平的干扰效果相同。结论采用混克隆细胞进行生物学特性鉴定和后续基因表达水平研究,实验信息量大、周期短、污染几率小、可重复性强,是一种省时、简便、高效的方法。Objective To compare the efficiency of gene interference between monoclonal and mixed clone cell during cell transfection.Methods Gastrin specific short/small hairpin RNAs （shRNAs）vector was transfected into L25 cell.RT-PCR, Western blot and immunofluorescence analyses were used for detecting gastrin expression at the mRNA level and protein level in mixed clone and monoclone which obtained by G418 resistence screening.Results The RT-PCR results showed that the most dramatic decrease in gastrin mRNA level was appeared in mixed clone. The gastrin expression level in 10 monoclones were discrepancy.Among the total monoelones, only pshRNA-1 and pshRNA-10 clones showed the significant decrease in gastrin mRNA level, and there interference efficiency was the same as mixed clone.Consistent with this found, Western blot and immunofluorescence analyses showed that in gastrin interference cells, pshRNA-mixed clone, pshRNA-1 and pshRNA-10 monoclones had almost the same gastrin expression efficiency.Conclusion In cell transfeetion experiments, there are a lot of problems on the selection of monoclones, such as long cycle for screening,the easiness of losing targeted gene in long-term preservation.While,directly using mixed clone for subsequent research give many advantages such as high authenticity of the experiment, being time-saving and less pollution risk, this method is superior to choose monoclones.

关 键 词：混克隆细胞 单克隆细胞 干扰效率 

分 类 号：R730[医药卫生—肿瘤]