Utilizing PB Transposon to Generate a Comprehensive Mouse Knockout Resource

利用 PB 转座子生成全面的小鼠敲除资源

• 批准号: 7488727
• 负责人: TIAN XU
• 金额: $ 5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-23 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7488727
• 关键词: Animal Housing; Animal Model; Animals; Biology; Breeding; Bypass; C57BL/6 Mouse; China; Classification; Communities; DNA Sequence Analysis; Databases; Disease; Disease model; Drug Industry; Embryo; Equipment and supply inventories; Freezing; Gene Expression; Generations; Genes; Genetic; Genetic Markers; Genomics; Health; Human; Human Biology; Indium; Insertional Mutagenesis; Knock-out; Knockout Mice; LacZ Genes; Mammals; Mediating; Methods; Modeling; Mus; Mutant Strains Mice; Mutate; Mutation; Numbers; Operative Surgical Procedures; Orthologous Gene; Pattern; Pharmacologic Substance; Phenotype; Play; Polymerase Chain Reaction; Process; Production; Reporter; Reporting; Research; Research Personnel; Resources; Role; Scheme; Siblings; Site; System; Techniques; Technology; Testing; Therapeutic; Time; Universities; Work; animal facility; comparative; cost; embryonic stem cell; gene function; genetic analysis; genetic technology; improved; mouse genome; mouse model; mutant; programs; response; sperm cell; success

The mouse shares 99% of the genes with humans and is an ideal research model for enhancing our understanding of human biology and disease and for testing potential therapeutics. Our ability to utilize the mouse model is limited by the number of mouse mutants and disease models (3000 - 4000) that are available to the research and pharmaceutical community. We have developed a new transposon system, called piggyBac (PB), which can be used to efficiently mutate a large number of genes in mice and other mammalian systems. This system has the following advantageous characters: 1. PB transposes efficiently in the mouse germline and can rapidly produce a large number of single PB insertion mutant strains; 2. Disrupted genes can be easily identified by PCR and sequencing; 3. PB insertions favor genes and have a wide genomic distribution; 4. PB insertions disrupt gene function when inserted into genes, and produce phenotypes similar to mutants generated by traditional knockout methods; 5. PB can carry large DMA fragments, e.g., an Act-RFP marker and a LacZ reporter, which can report gene expression patterns; 6. The resulting heterozygous or homozygous mutant animals and their wild-type siblings can be visually distiguished from each other without PCR or Southern using reporters such as Act-RFP; 7. Mutant animals are directly produced by simple breeding, which bypasses costly and challenging traditional techniques involving ES cells and surgery; 8. Visible genetic markers are employed in the breeding scheme to further improve efficiency and reduce cost; 9. All mutants are generated in an identical genetic background; 10. PB insertions can be precisely excised to revert the mutant phenotypes. We propose to utilize this PB insertional mutagenesis system and the cost-effective animal facility in Shanghai, China to produce 100,000 independent single PB insertion mutant strains in C57BL/6 mice in five years. We will determine the insertion sites, establish a database, and generate a comprehensive mouse KO resource which consists of frozen mutant embryos or sperm for approximately unique 20,000 genes or genetic loci. We estimate that the costs for the production and characterization of an independent insertion strain and for the storage of that strain are approximately $148 and $760, respectively. This comprehensive mouse mutant resource will significantly aid our ability to understand biology and improve human health.

小鼠与人类共有99%的基因，是增强我们的免疫力的理想研究模型。 了解人类生物学和疾病以及测试潜在的治疗方法。我们利用 小鼠模型受到小鼠突变体和疾病模型数量的限制（3000 - 4000）， 提供给研究和制药界。我们开发了一种新的转座子系统， piggyBac（PB），可用于有效地突变小鼠和其他动物的大量基因。 哺乳动物系统该系统具有以下优点：1. PB有效地在 小鼠种系，并能快速产生大量的单PB插入突变株; 2. 破坏的基因可以很容易地通过PCR和测序鉴定; 3. PB插入有利于基因， 广泛的基因组分布; 4. PB插入在插入到基因中时破坏基因功能，并产生 与通过传统敲除方法产生的突变体相似的表型; 5. PB可以携带大型DMA 片段，例如，Act-RFP标记和LacZ报告基因，其可以报告基因表达模式; 6.的 所得到的杂合或纯合突变动物及其野生型同胞可以在视觉上 在没有PCR或Southern的情况下使用诸如Act-RFP的报道基因彼此区分; 7.突变动物 通过简单的育种直接生产，绕过了昂贵且具有挑战性的传统技术 涉及胚胎干细胞和手术; 8.在育种方案中采用可见的遗传标记， 提高效率，降低成本; 9.所有突变体都是在相同的遗传背景下产生的; 10. PB 可以精确地切除插入以恢复突变表型。 我们建议利用这种PB插入突变系统和具有成本效益的动物设施， 中国上海，在五个C57 BL/6小鼠中生产100，000个独立的单PB插入突变株 年我们将确定插入位点，建立数据库，并生成全面的小鼠KO 由冷冻突变胚胎或精子组成的资源，约有20，000个独特的基因，或 遗传位点我们估计，生产和表征独立插入的成本 菌种和菌种储存费分别约为148美元和760美元。这一全面 小鼠突变资源将大大有助于我们理解生物学和改善人类健康的能力。