Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.
Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.
复制标题
基质附着区域组合增加转染的中国仓鼠卵巢细胞中的转基因表达
DOI：
10.1038/srep42805
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发表时间：
2017-02-20
影响因子：
4.6
通讯作者：
Wang TY
中科院分区：
文献类型：
--
作者：
Zhao CP;Guo X;Chen SJ;Li CZ;Yang Y;Zhang JH;Chen SN;Jia YL;Wang TY
基质附着区（MAR）是可以增加CHO细胞表达系统中的转基因表达水平的顺式作用DNA元件。为了研究MAR组合对转基因表达的影响和潜在的调控机制，我们产生了构建体，其中增强型绿色荧光蛋白（eGFP）基因侧翼为人β-干扰素和β-珠蛋白MAR（分别为iMAR和gMAR）的不同组合，其由巨细胞病毒（CMV）或猿猴病毒（SV）40启动子驱动。将这些转染到CHO-K1细胞中，用遗传霉素筛选;通过流式细胞术检测eGFP表达。MAR元件的存在增加了两种启动子下eGFP表达的转染效率和瞬时稳定表达;当两种MAR不同时（即，iMAR和gMAR），但不是SV 40启动子。对于后者，两个gMAR显示出最高的活性。我们还发现MAR增加了稳定转染的阳性集落的比例。这些结果表明，将CMV启动子与两个不同的MAR元件组合或将SV 40启动子与两个gMAR组合可有效诱导转基因的高表达水平和稳定性。
Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.