Transposon Based Mammalian Transgenesis and Transfection

描述(申请人提供)：用于哺乳动物转基因的转座子载体的开发有可能解决逆转录病毒(如慢病毒)载体的许多局限性，如有限的转基因大小和随机整合。我们最近已经证明，最初从卷心菜环纹夜蛾中分离出来的porgyBac转座子，在转座方面比曾被认为是最活跃的转座子的过度活跃的睡美人更有效。我们的第一个目标是探索单质粒转座酶-转座子(辅助者和供体)将转座子中的转基因酶插入宿主基因组染色体四核苷酸(TTAA)位点的可能性。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。已开发出几种制备转基因小鼠的方法，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)采用了一种主动的转基因插入模式，其他的依靠卵母细胞的修复机制进行转基因整合。自从ICSI-Tr只对冷冻解冻的精子起最佳作用以来，我们专注于通过开发积极的转基因程序来提高转基因在小鼠中的整合。在蛋白重组酶和转座酶的方法中，高活性的Tn5转座酶蛋白(*Tn5p)是目前为止将转座子中的转基因基因与未冻融精子一起导入未受精卵母细胞(TN：ICSI)的最有效方法。然而，由于TN：ICSI受制于繁琐的酶制备技术，我们现在已经不再使用酶插入转基因，而是开发了基于DNA的程序，允许原位合成转座酶。此外，为了达到靶向特异性，我们将GAL4 DNA结合域与iggyBac蛋白的N端进行了融合，并通过染色体整合实验确定了嵌合体的活性。GAL4-PiggyBac转座酶的活性与野生型PiggyBac相似，并插入到其常规的TTAA位点处。因此，我们认为这个转座子系统，由于其对分子工程的灵活性和相对较高的转座活性，可能是哺乳动物转基因和临床前基因治疗实验的理想选择。对于第一个特定的目的，我们还将利用与GAL-4DNA结合域偶联的嵌合转座酶，通过UAS串联阵列定向整合到小鼠胚胎基因组中。在第二个目标中，我们将开发连接到锌指DNA结合域的转座酶，以DNA和cRNA的形式识别酪氨酸酶基因座，以在细胞系和动物中进行靶向基因组整合。因此，这笔赠款将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。

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.