Endo-Porter–mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis

BioTechniques

Carissa M. Krane¹  Matthew V. Puccetti¹  Venkateshwar Mutyam¹  David L. Goldstein²  James Frisbie² 

[1] 1Department of Biology, University of Dayton, Dayton, OH, USA;2Department of Biological Sciences, Wright State University, Dayton, OH, USA;

关键词: aquaporin;    aquaglyceroporin;    Cope's gray treefrog;    morpholino-mediated knockdown;    Endo-Porter;    suspension cultures;   

DOI  :  10.2144/000113671

来源: DOAJ

【 摘 要 】

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).

【 授权许可】

Unknown