High Fidelity Linear MicroVector to Clone Complex, Problematic, and Large DNAs

用于克隆复杂、有问题和大型 DNA 的高保真线性微载体

• 批准号: 9346284
• 负责人: DAVID Alan MEAD
• 金额: $ 22.43万
• 依托单位: VARIGEN BIOSCIENCES CORPORATION
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9346284
• 关键词: Achievement; Bacteria; Bacteriophages; Basic Science; Cells; Cessation of life; Chromosomes, Human, 16-18; Cloning; Cloning Vectors; Complex; Coupled; Cytomegalovirus; DNA; DNA Library; DNA biosynthesis; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Diagnostic; Escherichia coli; Gene Proteins; Genes; Genetic; Genetic Research; Genome; Genomic DNA; Genomic Library; Genomic Segment; Genomics; Goals; Health; Human; Human Genome; Institutes; Letters; Libraries; Ligation; Maintenance; Malaria; Mammalian Cell; Methods; Nature; Nuclear; Nuclear Localization Signal; Parasites; Pathway interactions; Phase; Plasmodium; Plasmodium falciparum; Procedures; Production; Proteins; Provider; Recovery; Repetitive Sequence; Replication Origin; Replicon; Reporter; Reporting; Research; Resources; Rhamnose; Sampling; Stem Cell Research; System; Therapeutic; Transfection; Trust; Work; World Health Organization; arm; base; ds-DNA; expression vector; gene synthesis; gene therapy; human DNA; improved; in vivo; inducible gene expression; meetings; novel; pathogen; practical application; single molecule; synthetic construct; therapeutic protein; vector

The goal of this research is to dramatically improve the ability to clone and analyze large and unstable DNA fragments. We propose to develop a novel linear cloning vector to maximize stability of cloned DNA in the bacterial host. The vector will use the replication proteins of the phage Phi29 to achieve the highest accuracy of replication for all DNA inserts, including AT-rich, repetitive, or structurally unstable genes. The cloning method will be quick and easy for DNA of any size range (0-100 kb), only requiring ligation of small vector arms to the DNA, followed by transformation of bacteria. This system will surpass existing standards for stability, fidelity, and lack of bias, exceeding the limits of our previously developed pJAZZ linear cloning vector. Importantly, it will enable straightforward cloning of fosmid- or BAC-sized DNAs. The vector will have minimal bacterial sequences, providing significant benefits for mammalian cell transfection, therapeutic protein production, stem cell research, and ultimately gene therapy. The cloned DNAs will be bound to nuclear localization signals for enhanced delivery and expression in mammalian cells. This Phi29 vector is urgently needed to help discover treatments for human illness. For example, the genome of the human malaria parasite, Plasmodium falciparum, is among the most difficult genomes to clone, due to its repetitive, highly AT-rich nature. We will collaborate with the Wellcome Trust Sanger Institute to construct and sequence long-insert genomic libraries of Plasmodium falciparum. This unique resource is critically needed to enable genetic research on this deadly pathogen.

这项研究的目的是显着提高克隆和分析大而不稳定的能力 DNA片段。我们建议开发一种新型的线性克隆向量，以最大化克隆的稳定性 细菌宿主中的DNA。向量将使用噬菌体Phi29的复制蛋白来实现 所有DNA插入物的复制精度最高，包括富裕，重复或结构不稳定 基因。对于任何尺寸范围（0-100 kb）的DNA，克隆方法将很容易 将小矢量臂连接到DNA，然后将细菌转化。这个系统将 超过现有的稳定，忠诚和缺乏偏见的标准，超过了我们以前的限制 开发了Pjazz线性克隆向量。重要的是，它将实现fosmid-或 BAC大小的DNA。 载体将具有最小的细菌序列，为哺乳动物细胞带来显着益处 转染，治疗性蛋白质产生，干细胞研究以及最终基因治疗。这 克隆的DNA将与核定位信号绑定，以增强和表达 哺乳动物细胞。 迫切需要这种Phi29载体来帮助发现人类疾病的治疗方法。例如， 人类疟疾寄生虫的基因组，恶性疟原虫，是最困难的基因组之一 克隆，由于其重复性，高丰富的性质。我们将与Wellcome Trust Sanger合作 构建和序列长期植入恶性疟原虫的基因组文库的研究所。这个独特 需要进行至关重要的资源来实现对这种致命病原体的遗传研究。