title: Endo-Porter-mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis.
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).
authors.affiliation: Department of Biology, University of Dayton, Dayton, OH, USA.
authors.forename: Venkateshwar
authors.initials: V
authors.lastname: Mutyam
authors.forename: Matthew V
authors.initials: MV
authors.lastname: Puccetti
authors.forename: James
authors.initials: J
authors.lastname: Frisbie
authors.forename: David L
authors.initials: DL
authors.lastname: Goldstein
authors.forename: Carissa M
authors.initials: CM
authors.lastname: Krane
mesh_terms: D000818:Animals; D001001:Anura / Q000378:metabolism*; D051397:Aquaglyceroporins / Q000187:drug effects* / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D009025:Morpholines / Q000378:metabolism*; D060172:Morpholinos; D009841:Oligonucleotides / Q000008:administration & dosage / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000235:genetics; D010455:Peptides / Q000008:administration & dosage* / Q000235:genetics; D013696:Temperature
chemical_list: D051397:Aquaglyceroporins; C511217:Endo-Porter; D009025:Morpholines; D060172:Morpholinos; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010455:Peptides; D005990:Glycerol