Li Qin, Zhao Chun-peng, Wang Xiao-yin, Sun Qiu-li, Wang Tian-yun
Sichuan Da Xue Xue Bao Yi Xue Ban. 2016 Mar;47(2):189-91, 243.
To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector.
The MAR of β-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method.
The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence.
Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.
确定内含子方向对基质附着区域（MAR）表达载体所施加的转基因表达水平的影响。
通过PCR扩增β-珠蛋白的MAR，然后克隆到MAR表达载体中。用限制性内切酶消化内含子序列，以反向连接到MAR表达载体上，然后转染到中国仓鼠卵巢（CHO）细胞中。用G418筛选转染的稳定细胞。采用ELISA法分析氯霉素乙酰转移酶（CAT）基因的表达水平。
带有正向内含子或无MAR的两种表达载体的CHO细胞的转基因表达水平高于带有反向内含子表达载体的CHO细胞（P<0.05）。在存在反向内含子的情况下，MAR不能提高转基因表达。
内含子的不同方向可影响重组CHO细胞中的转基因表达。使用正向内含子和MAR可提高转基因表达水平。
