Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo
Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo
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DOI：
10.1038/gt.2009.109
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发表时间：
2010-01-01
期刊：
影响因子：
5.1
通讯作者：
Legrand, N.
中科院分区：
文献类型：
--
作者：
Centlivre, M.;Zhou, X.;Legrand, N.
从慢病毒载体体内有效控制基因表达仍然具有技术挑战性。为了分析人类环境中的诱导型基因表达，我们通过将用强力霉素诱导型慢病毒载体转导的人造血干细胞移植到新生BALB/c Rag 2（-/-）IL-2 R γ（-/-）（c）免疫缺陷小鼠中来产生“人免疫系统”（HIS）小鼠。我们比较了多西环素给药小鼠的几种方法，并可以准确地测量多西环素在体内使用一种新的灵敏的检测方法。使用具有优化的反向四环素反式激活因子的组成型（TRECMV-V14）或自动调节型（TREAuto-V14）表达的两种不同的慢病毒载体设计来扩增人造血干细胞。移植到免疫缺陷小鼠后，我们分析了绿色荧光蛋白（GFP）报告基因在人造血衍生细胞中的表达，这些细胞在生成的HIS小鼠中发育和积累。我们在含有TREAuto-V14转导的人细胞的成年HIS小鼠中显示了有效的诱导型GFP表达，而用TRECMV-V14载体GFP表达较差。TREAuto-V14组中多西环素暴露的多个循环导致GFP表达的重复循环，而没有强度损失。这些发现对于需要诱导基因表达的基因治疗和基础研究环境具有重大意义。Gene Therapy（2010）17，14-25; doi：10.1038/gt.2009.109; 2009年9月3日在线发表
The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-) IL-2R gamma(-/-)(c) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression. Gene Therapy (2010) 17, 14-25; doi: 10.1038/gt.2009.109; published online 3 September 2009