摘要
β干扰素基质附着区(MARs)的特征序列通过基因在中国仓鼠卵巢细胞(CHO)能介导的转基因载体表达。然而,宿主细胞是从仓鼠卵巢,这是不适合的靶细胞的基因治疗。在这项研究中,我们的目的是评估12种不同的人类细胞系作为基因治疗的靶细胞的适用性。转染的细胞类载体,获得了稳定表达载体产品G418筛选后的殖民地。因此,稳定转染的细胞分裂成了两个,进一步培养在存在或G418的情况下。流式细胞术观察细胞转染阳性率及绿色荧光蛋白(GFP)表达水平。质粒拯救试验,荧光原位杂交(FISH),荧光定量聚合酶链反应(PCR)被用来研究在哺乳动物细胞中的质粒的存在和基因拷贝数。结果表明,转染效率和转基因在A375,Eca-109水平,和ChangLiver细胞表达高。相反,观察BJ,HSF和A431细胞的转基因沉默,以及在其他六个细胞系观察到基因低表达。此外,该质粒在A375, Eca-109 和常肝细胞中游离状态存在,具有较低的拷贝数,即使在非选择性条件。因此,我们的研究结果提供了第一手的证据,关于基质附着区的特征序列介导的附加型载体的转基因表达对不同细胞类型的附加体长期稳定性的维护
关键词: 核基质附着区,Eposimal载体,外源基因的表达水平,宿主细胞
Current Gene Therapy
Title:Cell Compatibility of an Eposimal Vector Mediated by the Characteristic Motifs of Matrix Attachment Regions
Volume: 16 Issue: 4
Author(s): Tian-Yun Wang, Li Wang, Yu-Xin Yang, Chun-Peng Zhao, Yan-Long Jia, Qin Li, Jun-He Zhang, Yi-You Peng, Miao Wang, Hong-Yan Xu and Xiao-Yin Wang
Affiliation:
关键词: 核基质附着区,Eposimal载体,外源基因的表达水平,宿主细胞
摘要: The characteristic sequence of β-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.
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Tian-Yun Wang, Li Wang, Yu-Xin Yang, Chun-Peng Zhao, Yan-Long Jia, Qin Li, Jun-He Zhang, Yi-You Peng, Miao Wang, Hong-Yan Xu and Xiao-Yin Wang , Cell Compatibility of an Eposimal Vector Mediated by the Characteristic Motifs of Matrix Attachment Regions, Current Gene Therapy 2016; 16 (4) . https://dx.doi.org/10.2174/1566523216666161202160936
DOI https://dx.doi.org/10.2174/1566523216666161202160936 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
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