A chimeric HS4 insulator-scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells.
A chimeric HS4 insulator-scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells.
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嵌合 HS4 绝缘子支架附着区增强转染中国仓鼠卵巢细胞中的转基因表达
DOI：
10.1002/2211-5463.12335
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发表时间：
2017-12
期刊：
影响因子：
2.6
通讯作者：
Wang TY
中科院分区：
文献类型：
--
作者：
Chen SJ;Wang W;Zhang FY;Jia YL;Wang XY;Guo X;Chen SN;Gao JH;Wang TY
中国仓鼠卵巢（CHO）细胞是用于生产重组蛋白的最常用的表达系统之一，但经常遇到低水平的转基因表达和转基因沉默。表观遗传调控元件，如鸡β珠蛋白基因座控制区超敏位点4（HS 4）和支架/基质附着区（S/MAR）对转基因表达有积极影响。在本研究中，将嵌合HS 4-SAR克隆到真核载体中增强型绿色荧光蛋白（eGFP）表达盒的上游或下游，并将所得载体转染到CHO细胞中。流式细胞术检测eGFP。使用真实的实时定量PCR（qPCR）测定稳定转染细胞的拷贝数。用荧光原位杂交（FISH）检测载体在宿主细胞染色体上的状态。结果表明，与对照载体相比，位于表达盒下游的HS 4 ‐SAR可使eGFP表达增强4.83倍。转基因拷贝数和基因表达水平之间可能没有关系。HS 4 ‐SAR似乎不会改变转基因整合到宿主细胞染色体中或其在染色体中的位置。我们发现合成的嵌合HS 4-SAR在CHO细胞中积极增加转基因表达。
Chinese hamster ovary (CHO) cells are one of the most commonly used expression systems for the production of recombinant proteins but low levels of transgene expression and transgene silencing are frequently encountered. Epigenetic regulatory elements such as the chicken β‐globin locus control region hypersensitive site 4 (HS4) and scaffold/matrix attachment regions (S/MARs) have positive effects on transgene expression. In this study, a chimeric HS4‐SAR was cloned upstream or downstream of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, and the resulting vectors were transfected into CHO cells. eGFP was detected by flow cytometry. Real‐time quantitative PCR (qPCR) was used to determine copy numbers of the stably transfected cells. And fluorescence in situ hybridization (FISH) was used to detect the status of vector in the host cell chromosome. The results showed that HS4‐SAR positioned downstream of the expression cassette could enhance eGFP expression by 4.83‐fold compared with the control vector. There may not be a relationship between transgene copy number and gene expression level. HS4‐SAR did not appear to alter the integration of the transgene into the host cell chromosome or its position in the chromosome. We found a synthetic chimeric HS4‐SAR positively increased transgene expression in CHO cells.
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影响因子：
5.1
作者：
Papapetrou, EP;Ziros, PG;Athanassiadou, A
通讯作者：
Athanassiadou, A
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