PDF(Pubmed)
Abstract：
For diploid model organisms, the actual transgenesis processes require subsequent periods of transgene management, which are challenging in emerging model organisms due to the lack of suitable methodology. We used the red flour beetle Tribolium castaneum, a stored-grain pest, to perform a comprehensive functional evaluation of our AClashOfStrings (ACOS) and the combined AGameOfClones/AClashOfStrings (AGOC/ACOS) vector concepts, which use four clearly distinguishable markers to provide full visual control over up to two independent transgenes. We achieved comprehensive statistical validation of our approach by systematically creating seventeen novel single and double homozygous sublines intended for fluorescence live imaging, including several sublines in which the microtubule cytoskeleton is labeled. During the mating procedures, we genotyped more than 20,000 individuals in less than 80 working hours, which corresponds to about 10 to 15 s per individual. We also confirm the functionality of our combined concept in two double transgene special cases, i.e. integration of both transgenes in close proximity on the same chromosome and integration of one transgene on the X allosome. Finally, we discuss our vector concepts regarding performance, genotyping accuracy, throughput, resource saving potential, fluorescent protein choice, modularity, adaptation to other diploid model organisms and expansion capability.
摘要：
对于二倍体模式生物，实际的转基因过程需要后续的转基因管理，由于缺乏合适的方法，这在新兴的模式生物中是具有挑战性的。我们用了红面粉甲虫Triboliumcastaneum,一种储存谷物的害虫，对我们的AClashOfStrings（ACOS）和组合的AGameOfClones/AClashOfStrings（AGOC/ACOS）矢量概念进行全面的功能评估，它使用四个明显可区分的标记来提供对多达两个独立转基因的完全视觉控制。我们通过系统地创建17种用于荧光实时成像的新型单纯合和双纯合亚系，实现了对我们方法的全面统计验证。包括标记微管细胞骨架的几个亚系。在交配过程中，我们在不到80个工作小时的时间里对2万多人进行了基因分型，这相当于每个人大约10到15秒。我们还证实了我们的组合概念在两个双转基因特殊情况下的功能，即两个转基因在同一染色体上紧密接近的整合和一个转基因在X同种异体体上的整合。最后,我们讨论关于性能的向量概念，基因分型的准确性，吞吐量，资源节约潜力,荧光蛋白的选择，模块性，适应其他二倍体模式生物和扩展能力。
