Nikopoulos George N, Adams Tamara L, Adams Derek, Oxburgh Leif, Prudovsky Igor, Verdi Joseph M
Maine Medical Center Research Institute, Center for Molecular Medicine, Scarborough, Maine 04074, USA.
Biotechniques. 2008 Apr;44(4):547-9. doi: 10.2144/000112725.
Cellular interactions in development of the kidney are used as a model of reciprocal inductive events between epithelium and mesenchyme. Time- and labor-intensive methods have been developed to study this phenomenon. For example, in mice, the targeted disruption of genes in vivo has been used to modify the genetic program directing kidney development. However, gene targeting is a resource-intensive approach and alternative strategies for gene and protein modification in the kidney need to be developed. Herein, we have developed an efficient system for the delivery of antisense morpholino to alter normal protein expression. We describe the use of Endo-Porter to effectively deliver morpholinos to all parts and regions of the kidney explant. Also, we definitively show via confocal microscopy and Western blot analysis that the use of Endo-Porter in delivering antisense morpholinos is robust throughout the entire kidney explant, providing efficient suppression of protein expression. This method saves time and cost when compared with targeted disruption and is an improvement upon previous kidney organ culture methods.
肾脏发育过程中的细胞相互作用被用作上皮细胞与间充质之间相互诱导事件的模型。人们已经开发出耗时费力的方法来研究这一现象。例如,在小鼠中,体内基因的靶向破坏已被用于修改指导肾脏发育的遗传程序。然而,基因靶向是一种资源密集型方法,需要开发用于肾脏中基因和蛋白质修饰的替代策略。在此,我们开发了一种高效的反义吗啉代寡核苷酸递送系统,以改变正常蛋白质表达。我们描述了使用内吞转运体将吗啉代寡核苷酸有效地递送至肾脏外植体的所有部分和区域。此外,我们通过共聚焦显微镜和蛋白质印迹分析明确表明,在内吞转运体递送反义吗啉代寡核苷酸时,在整个肾脏外植体中效果良好,能有效抑制蛋白质表达。与靶向破坏相比,该方法节省时间和成本,并且是对先前肾脏器官培养方法的改进。