Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector
Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector
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DOI：
10.1073/pnas.230446297
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发表时间：
2000-11-21
影响因子：
11.1
通讯作者：
Clackson, T
中科院分区：
文献类型：
--
作者：
Pollock, R;Issner, R;Clackson, T
小分子调控转录具有广泛的用途，并且将受益于易于传递的自包含调控盒，能够强大，严格控制靶基因表达。我们描述了一种修饰的二聚体调控基因表达系统的传递到单个逆转录病毒上的细胞。通过将一个高效的嵌合激活域融合到fkbp -雷帕霉素相关蛋白（FRAP）的雷帕霉素结合域，优化了一种对天然产物二聚体雷帕霉素敏感的转录因子盒式转录病毒的递送。这种改善导致雷帕霉素或非免疫抑制性雷帕霉素类似物诱导的基因表达的效力和最大水平的增加。修饰后的转录因子盒与靶基因一起被整合到一个单一的雷帕霉素应答逆转录病毒中。用单病毒系统稳定转导的细胞池显示可忽略不计的基础表达，并且在雷帕霉素或非免疫抑制性雷帕霉素类似物存在下的诱导率至少为3个数量级。诱导的基因表达水平与组成型逆转录病毒长末端重复序列获得的水平相当，并且单个病毒系统在四种不同的哺乳动物细胞系中表现良好。二聚体反应性逆转录病毒的调控足够严格，可以产生可诱导表达高毒性白喉毒素A链基因的细胞系。在单个逆转录病毒中传递紧密诱导的雷帕霉素系统的能力将有助于其在广泛细胞类型的基因功能研究中的应用。
Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.