Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid.
Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid.
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DOI：
10.1186/scrt56
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发表时间：
2011-03-14
影响因子：
7.5
通讯作者：
Pestka S
中科院分区：
文献类型：
--
作者：
Krause CD;Izotova LS;Ren G;Yuan ZR;Shi Y;Chen CC;Ron Y;Pestka S
B16肿瘤内干扰素的局部合成介导抗肿瘤作用。基于干细胞被招募到肿瘤的报道，并且由于干扰素的全身给药引起剂量限制性的不良副作用，我们希望通过采用间充质干细胞（MSC）作为干扰素的肿瘤局部异位生产者来改善干扰素的抗肿瘤作用，同时最小化其全身副作用。存在许多载体来实现这一目的，但它们的转染效率和所得表达水平差异很大。为了跟踪MSC向肿瘤的募集和干扰素的合成，我们设计了一种双顺反子载体系统，其允许载体转染和产生干扰素的MSC的荧光可视化。我们用Mu-IFNαA cDNA作为第一顺反子，樱桃荧光蛋白cDNA作为第二顺反子，其翻译需要脑心肌炎病毒5'非翻译区的内部核糖体进入序列（IRES）。观察到这些顺反子在各种载体和细胞系中的不一致表达，特别是与对照质粒pmaxGFP相比，我们通过突变pcDNA 3以促进控制感兴趣的基因和真核抗生素抗性基因以及真核抗生素抗性基因本身的启动子和多聚腺苷酸化区段的交换来优化该双顺反子信息的表达，并有效地比较这些交换的效果，产生质粒pc3.5。稳定异位表达Mu-IFNαA的小鼠MSCs可抑制C57/BL 6小鼠移植瘤的形成。由双顺反子信使表达的Mu-IFNαA具有完全的生物活性，但表达水平仅为由单顺反子信使观察到的水平的三分之二。在293 T、B16和MSC细胞系中，帽依赖性翻译比IRES驱动的翻译效率高三倍。有效表达和良好转染效率都需要目的基因和嵌合内含子的强表达。肿瘤内高剂量的Mu-IFNαA抑制肿瘤建立，但可能不抑制肿瘤生长。我们的修饰载体及其衍生质粒将用于干细胞治疗、基因表达、mRNA调控和转录调控。肿瘤内Mu-IFNαA的局部释放可不同地影响肿瘤建立和肿瘤生长。
Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably. To follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5. Murine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth. Our modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.
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影响因子：
3.5
作者：
Bianchi, Marzia;Crinelli, Rita;Magnani, Mauro
通讯作者：
Magnani, Mauro
影响因子：
4.6
作者：
Amakata, Y;Fujiyama, Y;Bamba, T
通讯作者：
Bamba, T
DOI：
10.1073/pnas.96.26.14937
发表时间：
1999-12-21
影响因子：
11.1
作者：
Luo, MJ;Reed, R
通讯作者：
Reed, R
影响因子：
3.5
作者：
INOUE, H;NOJIMA, H;OKAYAMA, H
通讯作者：
OKAYAMA, H
影响因子：
6.2
作者：
Bartholomew, Amelia;Polchert, David;Kenyon, Norma
通讯作者：
Kenyon, Norma