Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells.
Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells.
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从人类基因组中鉴定有效的 MAR 元件并评估其在稳定转染的 CHO 细胞中的活性
DOI：
10.1111/jcmm.13361
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发表时间：
2018-03
影响因子：
5.3
通讯作者：
Xu GH
中科院分区：
文献类型：
--
作者：
Tian ZW;Xu DH;Wang TY;Wang XY;Xu HY;Zhao CP;Xu GH
低水平和不稳定的转基因表达是 CHO 细胞表达系统的常见问题。基质附着区（MAR）可增强转基因表达水平，但需要进行额外的研究来改善其功能并确定其作用机制。来自 CHO 染色体的 MAR-6 积极介导高且一致的基因表达。在这项研究中，我们比较了两种新的 MAR 和 MAR-6 对重组 CHO 细胞转基因表达的影响，发现了一种有效的 MAR 元件，可以显着增加转基因表达。克隆了两个 MAR，包括人 CSP-B MAR 元件和来自 CHO 细胞的 DHFR 内含子 MAR 元件，并将其插入真核载体中的 Poly(A) 位点下游。将构建物转染至CHO细胞中，并通过流式细胞术检测eGFP的表达水平和稳定性。三个 MAR 序列可以根据整体 eGFP 表达按降序排列，如下：人 CSP-B、DHFR 内含子 MAR 元件和 MAR-6。此外，正如预期的那样，与不含 MAR 的载体相比，三种含 MAR 的载体显示出更高的转染效率和瞬时转基因表达。生物信息学分析表明，MAR序列中的NFAT和VIBP元件可能有助于eGFP表达的增强。总之，人CSP-B MAR元件可以改善转基因表达，其作用可能与NFAT和VIBP元件有关。
Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.
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DOI：
10.1007/978-90-481-2245-5_1
发表时间：
2009-07-19
期刊：
Cell Line Development
影响因子：
--
作者：
Gorman C;Arope S;Grandjean M;Girod PA;Mermod N
通讯作者：
Mermod N
影响因子：
5.1
作者：
Papapetrou, EP;Ziros, PG;Athanassiadou, A
通讯作者：
Athanassiadou, A
影响因子：
5.3
作者：
Namciu, SJ;Blochlinger, KB;Fournier, REK
通讯作者：
Fournier, REK
影响因子：
5.3
作者：
PHIVAN, L;VONKRIES, JP;STRATLING, WH
通讯作者：
STRATLING, WH
影响因子：
56.9
作者：
BODE, J;KOHWI, Y;KOHWISHIGEMATSU, T
通讯作者：
KOHWISHIGEMATSU, T