Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System
Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System
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DOI：
10.1186/1475-2859-11-154
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发表时间：
2012-12-06
影响因子：
6.4
通讯作者：
Slavcev, Roderick
中科院分区：
文献类型：
--
作者：
Nafissi, Nafiseh;Slavcev, Roderick
背景：传统的非病毒基因传递DNA载体虽然比病毒载体更安全，但安全性有限。它们经常导致传递不需要的原核序列，抗生素抗性基因和细菌复制的起源到目标，这可能导致刺激不需要的免疫反应，由于它们的嵌合DNA组成。这些载体也可能赋予染色体整合的潜力，从而增强肿瘤的发生。我们试图设计一种体内系统，用于快速简单地生产更安全的DNA载体替代品，这种替代品不含非转基因细菌序列，并且在发生不需要的载体整合事件时，会致命地破坏宿主染色体。结果：我们构建了一个亲本真核表达载体，拥有一个专门制造的多靶点位点，称为“超级序列”，并工程大肠杆菌细胞（r细胞）有条件地产生噬菌体衍生的重组酶Tel (PY54), TelN （N15）或Cre （P1）。在优化的条件下，亲本质粒载体通过r细胞，可以快速、高效、一步地在体内生成与主质粒DNA分离的迷你lc -线性共价封闭（Tel/TelN-cell）或迷你cc-环状共价封闭（Cre-cell） DNA构建体。lcc质粒在宿主染色体上的位点特异性整合导致染色体断裂，其生存能力比ccc质粒低105倍。结论：我们提供了一种高效的迷你DNA载体生产系统，该系统提供了简单，快速和可扩展的迷你lcc DNA载体的体内生产，具有“迷你圆”DNA载体的所有优点，并且几乎消除了潜在的不良载体整合事件。
Background: While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event.Results: We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called "Super Sequence", and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc-linear covalently closed (Tel/TelN-cell), or mini ccc-circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart.Conclusion: We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of "minicircle" DNA vectors and virtually eliminate the potential for undesirable vector integration events.