Transgenic tools for the site specific insertion of large genomic transgenes via the PhiC31 integrase

转基因技术允许科学家探测基因功能，并通过产生携带特定感兴趣基因的额外拷贝的转基因小鼠品系来评估特定基因对特定生理过程的贡献。这项技术还允许建立人类遗传疾病和变异的模型，并通过将等效的人类突变引入小鼠进行研究。这些模型可用于调查疾病过程的潜在原因，并试验治疗和诊断方法。人类基因组序列的公布和最近的全基因组关联研究已经确定了许多功能尚未确定的基因和突变，因此预计转基因小鼠的使用将在未来几年急剧增加，以应对这些挑战。尽管这项技术的力量，目前使用的方法有相当显著的缺点，这使得技术相当不可预测。因此，遗传模型的生成和分析需要大量的工作、财政资源和动物研究。这种可预测性的缺乏通常是由于两个共同的弱点：首先，引入小鼠的遗传物质通常是基因的人工版本，缺乏许多潜在的调控域。因此，在小鼠体内产生的转基因表达确实能很准确地反映真实的生理情况。其次，添加到小鼠体内的遗传物质完全随机地进入小鼠染色体，可能造成损伤，并经常导致遗传物质的失调。再一次，转基因表达在这种情况下与生理无关。缺乏可预测性意味着从转基因模型中得出结论非常具有挑战性，并且必须生成和分析多个冗余菌株。此外，使用这种传统技术，很难比较携带不同突变的类似转基因的不同小鼠品系——随着我们解开DNA的自然变异，这一实验变得越来越重要。这种变异可能对疾病易感性产生重要影响，了解DNA中的这些微小差异如何与生理和疾病过程相关正成为一个重要的经常被问到的问题。该项目旨在通过开发工具来对抗这些技术的不利和不可预测的方面，这些工具可以将大区域的遗传物质（因此代表真正的基因而不是人工的迷你基因）整合到小鼠基因组的特定位置。整合的地点被选为中性；这意味着在这个位置插入遗传物质不会带来任何不良后果。将开发工具，使编码完整基因的大片段遗传信息能够被高效地操纵和转移到小鼠胚胎干细胞中。利用一种通常允许细菌病毒融入宿主基因组的酶，大的基因组片段将被插入基因组内的特定位置。由此产生的干细胞可用于产生携带这些转基因基因的转基因小鼠。对序列中的突变或变异的直接比较可以通过产生相同的转基因小鼠模型来进行，该模型在基因组的同一位置上携带所研究的基因序列的两个或多个版本。通过对小鼠的分析，我们可以很有把握地确定突变或变异的影响。

Transgenic technology allows scientists to probe gene function and to assess the contribution of specific genes to a particular physiological process through the generation of strains of genetically modified mice which carry extra copies of a particular gene of interest. This technology also allows models of human genetic disease and variation to be established and investigated by introducing equivalent human mutations into the mouse. These models can be used to investigate the underlying cause of the disease process and to trial therapeutic and diagnostic approaches. The publication of the human genome sequence and recent genome-wide association studies have identified many genes and mutations whose function has not yet been ascertained, thus it is expected that the use of genetically modified mice will increase dramatically over the coming years to address these challenges. Despite the power of this technology, the current methodologies in use have rather significant shortcomings which make the technology rather unpredictable. Consequently, a large amount of work, financial resources and animal studies are required for the generation and analysis of a genetic model. Frequently this lack of predictability is due to two common weaknesses: Firstly, the genetic material introduced into the mouse is frequently an artificial version of the gene, deficient in potentially many regulatory domains. Consequently the transgene expression which results in the mouse does very accurately reflect the real physiological situation. Secondly, the genetic material being added to the mouse enters the mouse chromosomes completely at random and can cause damage and frequently results in disregulation of the genetic material. Once again the transgene expression is not really physiologically relevant in this case. The lack of predictability means that it is very challenging to draw conclusions from transgenic models and multiple redundant strains must be generated and analysed. Furthermore, using this conventional technology, it is difficult to compare different strains of mice carrying similar transgenes with different mutations - an experiment which is becoming increasingly relevant as we unravel the natural variation in DNA. This variation may have important consequences for disease susceptibility and understanding how these small differences in DNA relate to physiological and disease processes is becoming an important frequently asked question. This project aims at combating these disadvantageous and unpredictable aspects of the technology by developing tools which enable large regions of genetic material (which thus represent real genes rather than artificial mini-genes) to be incorporated into specific sites within the mouse genome. The site of integration has been selected as being neutral; meaning that insertion of genetic material at this position is not associated with any undesirable consequences. Tools will be developed which enable large fragments of genetic information encoding for complete genes, to be manipulated and transferred into mouse embryonic stem cells at high efficiency. By taking advantage of an enzyme which normally allows a bacterial virus to integrate into its host's genome, large genomic fragments will be inserted in a specific site within the genome. The resulting stem cells can be used to generate strains of transgenic mice carrying these transgenes. A direct comparison of a mutation or variation in a sequence can be performed by generating identical transgenic mouse models which carry the two or more versions of the gene sequence understudy within the same position within the genome. By analysing the mice, we can ascertain the effects of the mutation or the variation with high confidence.