TRANSPOSON TARGETED INTEGRATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本申请的总体目标是开发生产转基因动物的新方法，最终重点是将在这些实验中学到的技术应用于基因治疗。为此，我们开发了一种新的主动转基因方法，比现有的被动方法有效数倍。该技术依赖于突变的超活性Tn 5转座酶蛋白（* Tn 5 p），用于在卵胞浆内单精子注射（ICSI）期间将转基因主动插入动物染色体。该方法通过显微注射和传统的ICSI-Transgenesis将外源DNA整合到动物基因组中，称为TN：ICSI。主动TN：ICSI技术的建立鼓励我们研究将Tn 5转座酶介导的转基因应用于基因治疗的方法。我们打算通过生产具有连接到 * Tn 5基因的DNA识别结构域的杂交蛋白来实现这一点。这将通过将嵌合转座子技术（CTT）的原理应用于杂交转座子构建体来实现，并尝试在啮齿动物中的转基因和基因治疗试验中使用这样的构建体。 基因治疗被定义为治疗通过患者的父母在受精过程中遗传的遗传疾病的手段。这类疾病在其遗传特征上往往是隐性的，并且仅在纯合子患者中表现出其全部影响。因此，发现两个杂合子父母都是没有症状的疾病携带者，而一个纯合子后代表现出这种疾病的显着表型并不罕见。基因治疗的目标是将一组健康的基因插入细胞中，在那里它可以取代一个有缺陷的版本，从而通过使用分子生物学技术来纠正这种遗传疾病。目前的基因治疗工作已经利用失活的慢病毒载体将治疗基因穿梭到患者细胞中，有时会带来灾难性的后果。在载体整合到患者基因组中期间引起的插入突变导致癌基因的激活，需要探索替代的活性基因插入方法。我们希望在转基因过程中定点插入 * Tn 5转基因将被证明是慢病毒技术的一个很好的替代方案。 PI已经开始与格鲁吉亚医学院（MCG）的Joseph Kaminski博士合作合成嵌合转座子（CTT）。 所述构建体将根据与其连接的DNA结合结构域（DBD）靶向感兴趣的DNA区域，用于转座子中包含的转基因的靶向整合。 最终的目标是影响整个动物的基因治疗。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this application is to develop novel methods for producing transgenic animals, with an eventual emphasis for the techniques learned during these experiments to be applied in gene therapy. To that effect we have developed a new active method of transgenesis that is several-fold more effective than the existing passive methods. The technique relies on a mutated hyperactive Tn5 transposase protein (*Tn5p) for the active insertion of a transgene into animal chromosomes during intracytoplasmic sperm injection (ICSI). This procedure named TN:ICSI integrates foreign DNA into the animal genome with microinjection and traditional ICSI-Transgenesis. The establishment of the active TN:ICSI technique has encouraged us to investigate methods for applying Tn5 transposase mediated transgenesis to gene therapy. We intend to accomplish this by the production of hybrid proteins with DNA recognition domains attached to the *Tn5 gene. This will be accomplished by applying principles of chimeric transposon technology (CTT) to a hybrid transposon construct and attempt to use such constructs in transgenesis and gene therapy trials in rodents. Gene therapy is defined as the means of curing genetic diseases which are inheritable via the patient's parents during fertilization. Such diseases tend to be recessive in their genetic signature and only exhibit their full effect in homozygous patients. Therefore, it is not unusual to find two heterozygous parents who are carriers for the disease with no symptoms, and a homozygous offspring who demonstrates a dramatic phenotype for the condition. The goal of gene therapy is to insert a healthy crop of a gene into a cell where it can take over for a faulty version and therefore correct such genetic diseases by using molecular biology techniques. Current gene therapy efforts have utilized deactivated Lentiviral vectors for shuttling therapeutic genes into patients cells, sometimes with disastrous consequences. The activation of oncogenes by insertional mutations caused during the vectors integration into the patients genome necessitates that alternative active gene insertion methods need to be explored. We hope that site directed *Tn5 transgene insertions during transgenesis will prove to be a good alternative to the Lentiviral technique. PI has started his collaboration with Dr. Joseph Kaminski of the Medical College of Georgia (MCG) for the synthesis of Chimeric Transposons (CTT). The constructs will target DNA regions of interest according to the DNA binding domains (DBD) attached to them, for targeted integration of transgenes contained in transposons. The eventual goal is to affect gene therapy in whole animals.