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.

这项研究的目标是显着提高克隆和分析大型和不稳定 DNA片段我们建议开发一种新的线性克隆载体，以最大限度地提高克隆的稳定性。 细菌宿主的DNA。该载体将使用噬菌体Phi 29的复制蛋白来实现 所有DNA插入片段的最高复制准确度，包括富含AT、重复或结构不稳定的插入片段 基因.该克隆方法对于任何大小范围（0-100 kb）的DNA都是快速和容易的，仅需要 将小载体臂与DNA连接，然后转化细菌。该系统将 超越现有的稳定性，保真度和无偏见的标准，超越我们以前的限制， 构建了pJAZZ线性克隆载体。重要的是，它将使直接克隆fosmid-或 BAC大小的DNA 该载体将具有最少的细菌序列，为哺乳动物细胞提供显著的益处。 转染、治疗性蛋白质生产、干细胞研究以及最终的基因治疗。的 克隆的DNA将与核定位信号结合，以增强在细胞中的递送和表达。 哺乳动物细胞 这种Phi 29载体迫切需要帮助发现人类疾病的治疗方法。比如说 人类疟疾寄生虫恶性疟原虫的基因组是最难研究的基因组之一。 克隆，由于其重复性，高AT丰富的性质。我们将与惠康信托桑格合作 构建恶性疟原虫长插入基因组文库并测序。这种独特 迫切需要资源，以便能够对这种致命的病原体进行遗传研究。