Utilizing PB Transposon to Generate a Comprehensive Mouse Knockout Resource

描述：（由申请人提供）小鼠与人类共享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社区可用的小鼠突变体和疾病模型（3000 - 4000）数量的限制。我们开发了一种新的转座子系统，称为pB，可用于有效地突变小鼠和其他哺乳动物系统中的大量基因。本系统具有以下优势特点：1。pB在小鼠种系中高效转座，能快速产生大量的单pB插入突变株；2 .通过PCR和测序可以很容易地鉴定出断裂的基因；3. pB插入有利于基因，具有广泛的基因组分布；4. 当插入基因时，pB插入会破坏基因功能，并产生与传统敲除方法产生的突变相似的表型；5. pB可携带较大的DNA片段，如Act-RFP标记和LacZ报告基因，可报告基因表达模式；6. 由此产生的杂合或纯合突变动物及其野生型兄弟姐妹可以在视觉上相互区分，而无需PCR或使用诸如Act-RFP之类的南方报告器；7. 通过简单的育种直接产生突变动物，这绕过了包括胚胎干细胞和手术在内的昂贵且具有挑战性的传统技术；8. 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. 可以精确地切除插入的pB以恢复突变表型。

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.