Document Type
Article
Publication Date
5-2011
Publication Source
BioTechniques
Abstract
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).
Inclusive pages
329–332
ISBN/ISSN
0736-6205
Document Version
Published Version
Copyright
Copyright © 2011, BioTechniques
Publisher
BioTechniques
Volume
50
Peer Reviewed
yes
Issue
5
eCommons Citation
Mutyam, Venkateshwar; Puccetti, Matthew V.; Frisbie, James; Goldstein, David L.; and Krane, Carissa M., "Endo-Porter–Mediated Delivery of Phosphorodiamidate Morpholino Oligos (PMOs) in Erythrocyte Suspension Cultures from Cope's Gray Treefrog Hyla Chrysoscelis" (2011). Biology Faculty Publications. 142.
https://ecommons.udayton.edu/bio_fac_pub/142
Included in
Biology Commons, Biotechnology Commons, Cell Biology Commons, Genetics Commons, Microbiology Commons, Molecular Genetics Commons
Comments
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