Utilizing PB Transposon to Generate a Comprehensive Mouse Knockout Resource

产品说明：小鼠与人类共有99%的基因，是增强我们对人类生物学和疾病的理解以及测试潜在治疗方法的理想研究模型。我们利用小鼠模型的能力受到研究和制药界可用的小鼠突变体和疾病模型数量（3000 - 4000）的限制。我们已经开发了一种新的转座子系统，称为pB，它可以用来有效地突变小鼠和其他哺乳动物系统中的大量基因。该系统具有以下优点：1. pB在小鼠生殖系中高效转座，并能快速产生大量单pB插入突变株; 2.通过PCR和测序可以很容易地鉴定出被破坏的基因; 3. pB插入有利于基因，具有广泛的基因组分布; 4. pB插入在插入基因时破坏基因功能，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可以携带大的DNA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.所得杂合或纯合突变动物及其野生型同胞可以在没有PCR或Southern的情况下使用报告基因如Act-RFP在视觉上彼此区分; 7.通过简单的育种直接产生突变动物，这绕过了涉及ES细胞和手术的昂贵且具有挑战性的传统技术;在育种方案中采用可见遗传标记，进一步提高效率，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.可以精确地切除pB插入以恢复突变体表型。 我们计划利用这个pB插入突变系统和中国上海的低成本动物设施，在5年内在C57 BL/6小鼠中产生100，000个独立的单pB插入突变株。我们将确定插入位点，建立数据库，并产生一个全面的小鼠KO资源，其中包括约20，000个独特基因或遗传位点的冷冻突变胚胎或精子。我们估计，一个独立的插入菌株的生产和表征以及该菌株的储存成本分别约为148美元和760美元。这种全面的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。

DESCRIPTION: (provided by applicant) 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 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 DNA 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 distinguished 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 20,000 unique 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.