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Rana catesbeiana Oocytes: An Alternative To The Xenopus laevis Heterologous Expression System For Ammonia Transporters
Author(s) -
Kabutomori Jessica,
BelotoSilva Olivia,
Geyer RR,
MusaAziz Raif
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.809.22
Subject(s) - xenopus , rana , oocyte , biology , aquaporin , salientia , bullfrog , microbiology and biotechnology , biochemistry , anatomy , endocrinology , embryo , gene
Xenopus laevis oocytes are a valuable tool for understanding the transport of H 2 O, CO 2 and/or ammonia (NH 3 /NH 4 + ) through mammalian aquaporins (AQPs), the glycosylated Rhesus (Rh) proteins (AG, BG and CG) and the urea transporter UT‐B, membrane proteins expressed in tissues involved in ammonium homeostasis and/or transport, such as the liver and the kidney. This is the actual focus of our research; however, regulations in Brazil render the import of Xenopus laevis frogs impractical. Here we evaluate the usefulness of North American aquatic bullfrog Rana catesbeiana , which is freely commercialized in Brazil, as an alternative. We have developed a collagenase‐based method for isolating individual defolliculated oocytes from Rana ovaries. We find that they are a similar size to Xenopus oocytes and are amenable to injection with cRNA, surface biotinylation protocols, western blot analysis, and measurements of osmotic water permeability (P f ) using a video camera to monitor the projection area of the oocyte as we reduce extracellular osmolality. Up to now our blotting data show that Rana oocytes are competent for plasma membrane expression of AQPs (1, 3, 5, and 9), RhAG, BG, and CG, and UT‐B, UT‐A2 and UT‐A3. Preliminary P f values for AQP1 (0.0018 ± 0.0003, n=5) and AQP5 (0.0022 ± 0.0005, n=5)‐injected Rana oocytes were ~10‐fold greater than for H 2 O (0.00024 ± 0.00004, n=5)‐injected control Rana oocytes, similar to historical values gathered from Xenopus oocytes. Thus we consider Rana oocytes to be a readily accessible system for the functional expression and study of membrane proteins for Brazilian researchers who do not currently have access to such a robust research tool.