Gillen Austin E, Lucas Catherine A, Haussecker Pei Ling, Kosak Steven T, Harris Ann
Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL, USA.
Chromosoma. 2013 Oct;122(5):351-61. doi: 10.1007/s00412-013-0418-9. Epub 2013 Jun 9.
Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.
细菌人工染色体（BAC）广泛应用于转基因研究，尤其是在动物模型人源化方面。此外，由于其容量大，BAC为研究与大基因座相关的远端调控元件提供了有吸引力的工具。然而，尽管它们被广泛使用，但对于这些大转基因在哺乳动物细胞中的整合动态却知之甚少。在这里，我们研究了通过细菌侵染递送携带囊性纤维化跨膜传导调节因子（CFTR）基因座的约260 kb BAC的整合后结构，并将其与更常规的基于脂质的递送方法的结果进行比较。我们发现细菌侵染介导的递送后，BAC CFTR转基因的整合拷贝数和表达水平存在很大差异。此外，我们经常观察到单个细胞克隆内CFTR转基因不同区域的呈现存在差异，这表明BAC发生了片段化。最后，使用荧光原位杂交，我们观察到整合的BAC在一些克隆中形成了延伸的兆碱基规模结构，这些结构在细胞分裂时显然能稳定维持。这些数据表明，在基因组背景下使用大BAC研究顺式调控元件的效用可能会受到使其使用复杂化的重组事件的限制。
