Transposon Based Mammalian Transgenesis and Transfection

基于转座子的哺乳动物转基因和转染

• 批准号: 8069618
• 负责人: STEFAN MOISYADI
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069618
• 关键词: Animals; Area; Binding; Biological Assay; Cabbage - dietary; Cell Line; Cell Nucleus; Cells; Chimera organism; Chromosomes; Color; Coupled; DNA; DNA Binding Domain; Development; Effectiveness; Embryo; Engineering; Enzymes; Exhibits; Freezing; Gene Delivery; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Engineering; Genome; Genomics; Goals; Grant; Human; Immune response; In Situ; Intracytoplasmic Sperm Injections; Knockout Mice; Lead; Lentivirus Vector; Location; Mammalian Cell; Mediating; Mediator of activation protein; Medical Research; Methods; Microinjections; Molecular; Monophenol Monooxygenase; Moths; Mus; Mutate; N-terminal; One-Step dentin bonding system; Oocytes; Organism; Planarians; Plasmids; Preparation; Procedures; Production; Proteins; Rec A Recombinases; Rodent Model; Site; Sleeping Beauty; Specificity; System; Techniques; Testing; Tn5 transposase; Transfection; Transgenes; Transgenic Animals; Transgenic Mice; Transposase; Virus; Work; Zinc Fingers; base; design; embryonic stem cell; field study; flexibility; gene therapy; human disease; improved; in vivo; method development; novel; pre-clinical; recombinase; repaired; research study; site-specific integration; sperm cell; technique development; vector; zygote

DESCRIPTION (provided by applicant): The development of transposon vectors for mammalian transgenesis has the potential to solve many of the limitations of retroviral (e.g., Lentiviral) vectors such as limited transgene size and random integration. We have recently demonstrated that the piggyBac transposon, originally isolated from the cabbage looper moth Trichoplusia ni, is more efficient at transposition than hyperactive Sleeping Beauty, once thought to be the most active transposon. Our first aim explores the possibility of a single plasmid transposase-transposons (Helper and Donor) ability to enzymatically insert a transgene in the transposon into the hosts genomic chromosome, at the teranucleotide (TTAA) site. We intend to use the information gained during cell transfection for the production of transgenic mice. Several methods have been developed for producing transgenic mice, including pronuclear microinjection, ICSI-Tr, virus-mediated insertion, and ES cell-mediated approaches. Of these only the virus-mediated insertion (Lentiviral) employs an active mode of transgene insertion, with the others relying on the repair mechanism of the oocyte for transgene integration. Since the development of ICSI-Tr which works optimally only with freeze-thawed sperm, we concentrated on improving the integration of transgenes in mice by developing active transgenesis procedures. Among approaches with protein recombinases and transposases, the hyperactive Tn5 transposase protein (*Tn5p) was by far the most efficient method when introducing the transgene in a transposon along with non freeze-thawed spermatozoa into unfertilized oocytes (TN:ICSI). However, because TN:ICSI suffers from cumbersome enzyme preparation techniques, we have now moved away from the enzymatic insertions of transgenes and developed DNA based procedures that allow synthesis of the transposase in-situ. Furthermore, to achieve target specificity, we fused the GAL4 DNA binding domain to the N-terminal of the piggyBac protein and determined the activity of the chimeras by chromosome integration assays. The GAL4-piggyBac transposase displayed an activity similar to that of wild-type piggyBac and inserted at its regular TTAA site. We therefore suggest that this transposon system, because of its flexibility for molecular engineering and its relatively high transposition activity, could be ideal for mammalian transgenesis and pre-clinical gene therapy experiments. For the first specific aim, we will additionally utilize a chimeric transposase coupled to the GAL-4 DNA binding domain for targeted integration into a mouse embryos genome with a UAS tandem array. In the second aim, we will develop transposases coupled to a zinc finger DNA binding domain, both in DNA and cRNA form, that recognize the tyrosinase locus for targeted genomic integration in both cell lines and animals. Therefore, this grant should lead to improved non-viral integrating vectors for use in mammalian transgenesis and animal gene therapy experiments.

描述（由申请人提供）：用于哺乳动物转基因的转座子载体的开发具有解决逆转录病毒的许多局限性的潜力（例如，慢病毒）载体，如有限的转基因大小和随机整合。我们最近已经证明，piggyBac转座子，最初从甘蓝夜蛾Trichoplusia ni分离，是更有效的转座比过度活跃的睡美人，曾经被认为是最活跃的转座子。我们的第一个目标是探索单个质粒转座酶-转座子（辅助和供体）在转座子中将转基因酶促插入宿主基因组染色体的三核苷酸（TTAA）位点的能力的可能性。我们打算利用细胞转染过程中获得的信息来生产转基因小鼠。已经开发了几种用于产生转基因小鼠的方法，包括原核显微注射、ICSI-Tr、病毒介导的插入和ES细胞介导的方法。其中，只有病毒介导的插入（慢病毒）采用转基因插入的主动模式，其他依赖于卵母细胞的修复机制进行转基因整合。由于ICSI-Tr的发展，它只与冻融精子最佳的工作，我们集中在改善小鼠的转基因整合，通过开发主动转基因程序。在使用蛋白质重组酶和转座酶的方法中，高活性Tn 5转座酶蛋白（* Tn 5 p）是迄今为止将转基因导入转座子中的最有效的方法，沿着非冻融精子进入未受精卵母细胞（TN：ICSI）。然而，由于TN：ICSI受到繁琐的酶制备技术的困扰，我们现在已经远离转基因的酶促插入，并开发了允许原位合成转座酶的基于DNA的程序。此外，为了实现靶特异性，我们将GAL 4 DNA结合结构域融合到piggyBac蛋白的N-末端，并通过染色体整合测定确定嵌合体的活性。GAL 4-piggyBac转座酶显示出与野生型piggyBac相似的活性，并插入其常规TTAA位点。因此，我们建议，这种转座子系统，由于其分子工程的灵活性和相对较高的转座活性，可能是理想的哺乳动物转基因和临床前基因治疗实验。对于第一个具体目标，我们将另外利用与GAL-4 DNA结合结构域偶联的嵌合转座酶，用于用UAS串联阵列靶向整合到小鼠胚胎基因组中。在第二个目标中，我们将开发与DNA和cRNA形式的锌指DNA结合域偶联的转座酶，这些转座酶识别酪氨酸酶位点，以在细胞系和动物中进行靶向基因组整合。因此，这笔赠款应导致改进的非病毒整合载体用于哺乳动物转基因和动物基因治疗实验。