Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression -: art. no. e106
Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression -: art. no. e106
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DOI：
10.1093/nar/gnh104
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发表时间：
2004-07-01
影响因子：
14.9
通讯作者：
Fussenegger, M
中科院分区：
文献类型：
--
作者：
Mitta, B;Weber, CC;Fussenegger, M
可调节的转基因表达被认为是基因治疗方案中下一代分子干预、组织工程临床细胞表型的治疗重编程以及后基因组时代复杂的基因功能分析的关键。我们设计了一系列最新一代自失活人类（HIV 衍生）和非人类（基于 EIAV）慢病毒表达载体，用于在各种啮齿类动物（CHO-K1、C2C12）和人类细胞系中链霉素调节报告基因（AMYS(DeltaS)、EYFP、SAMY、SEAP）、分化调节（人 C/EBP-a）和治疗性（人 VEGF）转基因表达(HT-1080、K-562)、人和小鼠原代细胞（NHDF、PBMC、CD4(+)）以及鸡胚胎。慢病毒设计概念包括（i）在单独的慢载体上包含组成型链霉素依赖性反式激活因子（PIT）和 PIT 响应转基因表达单元的二元系统； (ii) 链霉素反应启动子 (P-PIR8) 置于所需转基因的 5' 端； (iii) 在修饰的无增强子 3' 长末端重复序列内； (iv) 双向自动调节配置，以慢病毒单载体形式提供链霉素响应性转基因表达。严格的定量分析揭示了基于 HIV 的直接 P-PIR 转基因配置，可为以下方面提供最佳调节性能：（I）细胞内和分泌产物蛋白的可调节表达，（ii）调节肌肉前体细胞系向脂肪细胞或成骨细胞的差异分化，以及（iii）鸡胚胎中的条件性血管化微调。通过将链阳菌素反应性转基因表达设计成自动调节的单载体格式，可以实现类似的性能。配备可调节转录调节选项的强大转导系统有望极大地推进临床和/或生物技术相关原代细胞和细胞系中复杂的分子干预。
Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AMYS(DeltaS), EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-a) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4(+)) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (P-PIR8) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct P-PIR-transgene configurations to provide optimal regulation performance for (I) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular Interventions in clinically and/or biotechnologically relevant primary cells and cell lines.