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首页> 美国卫生研究院文献>Nucleic Acids Research >Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression
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Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression

机译：设计用于链霉菌素调节的转基因表达的自灭活人类和非人类慢病毒表达载体的设计和体内表征

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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 (AmySΔS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-α) 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 (PPIR8) 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 PPIR-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.
机译：可调节的转基因表达被认为是基因治疗场景中的下一代分子干预，用于组织工程的临床细胞表型的治疗性重编程以及后基因组时代复杂的基因功能分析的关键。我们设计了最新一代自我灭活的人类（HIV衍生）和非人类（基于EIAV）慢病毒表达载体，这些载体经工程设计用于链霉菌素可调节的报告基因（AmySΔS，EYFP，SAMY，SEAP）的表达， （人类C /EBP-α）和治疗性（人类VEGF）转基因在各种啮齿动物（CHO-K1，C2C12）和人类细胞系（HT-1080，K-562），人类和小鼠原代细胞（NHDF，PBMC）中，CD4 + ）以及鸡胚。慢病毒设计概念包括：（i）在独立的慢载体上包含组成型链霉菌素依赖性反式激活因子（PIT）和PIT响应性转基因表达单元的二元系统； （ii）放置在所需转基因5'端的链霉素反应性启动子（PPIR8）； （iii）在修饰的无增强子的3'长末端重复序列内； （iv）双向自动调节的配置，可提供慢病毒单载体格式的链霉素响应基因表达。严格的定量分析显示，基于HIV的直接PPIR转基因配置可为（i）细胞内和分泌产物蛋白的可调节表达，（ii）肌肉前体细胞系分化为脂肪细胞或成骨细胞的分化分化和（iii）有条件的分化提供最佳调节性能。鸡胚的血管化微调。通过将链蛋白反应性转基因表达工程化为一种自动调节的单载体格式，可以实现类似的性能。配备有可调节的转录调制选项的功能强大的转导系统有望在临床和/或生物技术相关的原代细胞和细胞系中大大促进复杂的分子干预。

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