Transient gene expression by nonintegrating lentiviral vectors
Transient gene expression by nonintegrating lentiviral vectors
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DOI：
10.1016/j.ymthe.2006.01.008
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发表时间：
2006-06-01
影响因子：
12.4
通讯作者：
Kohn, Donald B.
中科院分区：
文献类型：
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作者：
Nightingale, Sarah J.;Hollis, Roger P.;Kohn, Donald B.
非整合性慢病毒（NIL）载体是由HIV-1为基础的慢病毒载体，通过引入突变的组合，使整合酶蛋白本身失活，并改变病毒LTR中的整合酶识别序列（att）。使用具有这些新的突变组合的NIL载体来扩增人T淋巴细胞系Jurkat和原代人CD 34（+）造血祖细胞，以通过增强型绿色荧光蛋白（eCFP）报告基因的瞬时表达来评估它们的功效。大多数失能的NIL载体导致初始高水平的eGFP表达（类似于90%的细胞），但表达是瞬时的，在不到1个月内朝向背景（< 0.5%）减少。转导的Jurkat细胞的Southern印迹分析证实了1个月时可检测的NIL载体序列（线性形式和一个和两个LTR环）的损失。尽管有工程化改变的任何组合，NIL载体的整合残留水平低（与野生型载体相比降低约10（4）倍）。基于对来自载体LTR和细胞染色体的接合处的DNA序列的分析，这些罕见的整合的NIL载体序列不是由整合酶驱动的机制介导的，这是由于工程化突变的逆转，而是更可能由背景重组事件产生。NIL载体的开发为在原代干细胞、造血细胞和淋巴细胞中高效瞬时表达基因提供了新的工具。
Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eCFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (similar to 90% of cells), but expression was transient, diminishing toward background (< 0.5%) within less than I month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.