Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.
Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.
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DOI：
10.1093/nar/23.21.4451
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发表时间：
1995
影响因子：
14.9
通讯作者：
J. Bergemann;K. Kühlcke;B. Fehse;I. Ratz;W. Ostertag;H. Lother
中科院分区：
文献类型：
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作者：
J. Bergemann;K. Kühlcke;B. Fehse;I. Ratz;W. Ostertag;H. Lother
用于基因转移和基因治疗的载体结合了整合酶和重组酶系统的优点。这通过将用于特异性DNA切除的两个loxP位点插入基于MESV的逆转录病毒载体中来实现。我们表明，这种“逆转录病毒lox系统”允许感染细胞和表达转移的基因。此外，我们构建了一个有效的逆转录病毒为基础的表达系统的修改Cre重组酶。通过使用阴性选择标记基因（胸苷激酶）进行从整合的逆转录病毒lox载体中切除DNA的功能试验。在用逆转录病毒lox载体感染的细胞中表达Cre，随后对其中发生位点特异性重组的细胞进行BrdU选择，导致大量独立的细胞克隆。这些结果通过详细的分子分析得到证实。此外，我们开发了逆转录病毒自杀载体，其中两个LTR的增强子/启动子元件被替换为lox序列。我们发现，位于逆转录病毒载体的LTR中的lox序列在逆转录病毒复制过程中是稳定的。该系统的潜在应用将是通过控制切除几乎完整的前病毒DNA来建立逆转录病毒感染细胞的回复突变体。
Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.