• 站内检索
    高级检索
  • 最新录用
    更多
文章摘要
CHO细胞Kcmf1基因内定点整合ZsGreen1报告基因的表达稳定性研究
Study on the Expression Stability of ZsGreen1 Reporter Gene in the Kcmf1 Gene of CHO Cell
投稿时间:2019-11-22  修订日期:2020-01-14
DOI:
中文关键词: 定点整合  稳定性  ZsGreen1
英文关键词: site-specific integration  stability  ZsGreen1
基金项目:国家高技术研究发展计划(863计划)(2015AA020802)
作者单位E-mail
杨 蕾 江南大学生物工程学院 yangleier@126.com 
丁学峰 江南大学生物工程学院  
蔡燕飞 江南大学药学院  
陈蕴 江南大学药学院  
龚笑海 江南大学药学院  
段作营 江南大学生物工程学院  
李华钟 江南大学生物工程学院  
金 坚 江南大学药学院 jianjin@jiangnan.edu.cn 
摘要点击次数: 15
全文下载次数: 0
中文摘要:
      以Kcmf1基因NW_003614172.1第629890碱基处定点整合ZsGreen1报告基因的CHO-K1细胞(2C3)为研究材料,采用倒置荧光显微镜和流式细胞仪定量分析ZsGreen1的表达,开展系统的稳定性表达研究。2C3细胞贴壁培养连续传代50代次,100%的细胞可以稳定表达ZsGreen1报告基因。无血清悬浮驯化培养2C3细胞后,细胞表达ZsGreen1的平均荧光强度明显降低;连续悬浮传代培养60代次,表达ZsGreen1的细胞数目显著增多,添加10%FBS到悬浮培养的细胞池,ZsGreen1阳性细胞比例上升到95%以上。流式细胞术分选细胞池中高、低表达ZsGreen1蛋白的2C3细胞,PCR确认它们均含有ZsGreen1报告基因;Q-PCR发现在高、低表达ZsGreen1蛋白的细胞中,相关的ZsGreen1 mRNA水平有明显的差异。提示CHO-K1细胞Kcmf1基因内非编码区域定点整合外源基因,不会因细胞分裂和生长而丢失外源表达基因;悬浮驯化需要优化培养基和培育条件,达到构建工程细胞的需求。
英文摘要:
      The CHO-K1 cells (2C3) with ZsGreen1 reporter gene integrated at the 629890 base of Kcmf1 gene NW_003614172.1 were used as the study material, and the expression of ZsGreen1 was quantitatively analyzed by inverted fluorescence microscope and flow cytometry, so as to carry out systematic stability expression study. The ZsGreen1 reporter gene could be expressed stably in 100% of 2C3 cells after continuous passage of 50 generations in adherent culture. After cultured without serum, the average fluorescence intensity of ZsGreen1 expression in 2C3 cells decreased significantly. After continuous suspension culture for 60 generations, the number of cells expressing ZsGreen1 increased significantly. When 10%FBS was added to the cell pool of suspension culture, the proportion of ZsGreen1 positive cells increased to over 95%. 2C3 cells with high and low expression of ZsGreen1 protein in the cell pool were sorted by flow cytometry, and PCR confirmed that they all contained ZsGreen1 reporter gene. Q-PCR found that there were significant differences in the level of related ZsGreen1 mRNA in cells with high and low expression of ZsGreen1 protein. It was suggested that the non-coding region of Kcmf1 gene in CHO-K1 cell integrated exogenous genes at specific site, and the exogenous expressed genes were not lost due to cell division and growth. The suspension and acclimation requires optimization of culture medium and cultivation conditions to meet the needs of constructing engineering cells.
View Fulltext   查看/发表评论  下载PDF阅读器