Department of Biology, University of Dayton, Dayton, OH, USA.
Biotechniques. 2011 May;50(5):329-32. doi: 10.2144/000113671.
Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).
高蟾氏树蛙（Hyla chrysoscelis）是一种耐寒的两栖动物，在冷驯化过程中会积累抗冻蛋白甘油。H. chrysoscelis 的红细胞表达水甘油通道蛋白 HC-3，这有助于甘油和水的跨膜转运。水甘油通道蛋白没有药理学抑制剂，目前也没有针对 H. chrysoscelis 的基因敲除工具。因此，我们采用了一种基于磷酰胺二酯修饰的吗啉代寡核苷酸（PMO）介导的表达敲低方法，为研究 HC-3 的功能提供了一个模型。我们描述了一种优化的新型程序，用于在非哺乳动物生理温度下培养的两栖动物红细胞悬浮液中特异性、高效地敲低 HC-3 的表达，该程序使用了 Endo-Porter。我们的方案包括三个关键步骤：37°C 下预孵育、在~23°C 下进行两轮 Endo-Porter 和 HC-3 PMO 给药以及 190 rpm 的连续晃动。这一系列步骤导致 HC-3 蛋白表达减少了 94%（Western blot），超过 65%的红细胞中 HC-3 的表达显著降低，另外 30%的细胞中几乎检测不到表达（免疫细胞化学）。
