Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells
Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells
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DOI：
10.1128/jvi.72.5.3720-3728.1998
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发表时间：
1998-05-01
影响因子：
5.4
通讯作者：
Plavec, I
中科院分区：
文献类型：
--
作者：
Agarwal, M;Austin, TW;Plavec, I
我们研究了逆转录病毒转基因在原代人淋巴细胞中的表达。我们的数据表明，在激活的原代CD4(+)T细胞中，转基因表达很高，但在有丝分裂静止的细胞中，转基因表达显著减少。将人β干扰素基因支架附着区(SAR)的DNA片段整合到载体中可以改善转基因表达，特别是在静止细胞中。SAR元件以定向依赖的方式发挥作用，并增强了Moloney小鼠白血病病毒和基于小鼠胚胎干细胞的载体的表达。对转导的T细胞的克隆分析表明，该SAR序列不允许转基因在位置无关的情况下表达，而是在细胞静止时阻止了表达的下降。在SCID-Hu胸腺-肝脏模型中，该SAR序列还增强了从逆转录病毒转导的CD34富集造血祖干细胞产生的T细胞中的转基因表达。我们使用含SAR的逆转录病毒载体表达了人类免疫缺陷病毒1型(HIV-1)Rev基因的反式显性突变体RevM10基因。与标准逆转录病毒载体相比，含有SAR的载体在体外抑制HIV-1病毒在受感染的CD4(+)外周血淋巴细胞群体中的复制效率高达2个数量级。这是首次证明SAR元件可用于改善逆转录病毒载体在人类原代T细胞中的表达。
We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4(+) T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4(+) peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.