Erythroid-specific inactivation of Slc12a6/Kcc3 by EpoR promoter-driven Cre expression reduces K-Cl cotransport activity in mouse erythrocytes

• Main Authors: Alper, S.L (Author), Brugnara, C. (Author), Dlott, J.S (Author), Hsu, A. (Author), Michael Snyder, L. (Author), Nishimura, K. (Author), Rivera, A. (Author), Shmukler, B.E (Author), Wohlgemuth, J. (Author)
• Format: Article
• Language: English
• Published: American Physiological Society 2022
• Subjects: adult; allele; animal cell; animal experiment; animal model; Article; atomic absorption spectrometry; centrifugation; controlled study; cre recombinase; cytoskeleton; erythroblast; erythrocyte; erythropoietin receptor; gene; gene expression; genetic transcription; genotype; germ line; hematocrit; hormone; ion transport; LoxP site; megalocytosis; membrane protein; mouse; mutation; nonhuman; peptide; peptide hormone; potassium chloride cotransporter; progeny; protein expression; protein function; protein tyrosine kinase inhibitor; radioisotopic flux; recombinase; red blood cell; reticulocytosis; serine; signal transduction; sodium chloride; sodium transport; staurosporine; threonine; tissue-specific knockout; transcription factor; transgene; urea; weaning
• Online Access: View Fulltext in Publisher

 Investigation of erythrocytes from spontaneous or engineered germ-line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss-of-function is embryonic-lethal, perinatal-lethal, or confers reduced post-weaning viability. Promoter regions of erythroid-specific genes have been used to engineer erythroid-specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid-specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR-Cre;Kcc3f/f mice, designed to express erythroid-specific knockout of the KCC3 K-Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K-Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3−/− germline knockouts. We demonstrate roughly proportionate preservation of K-Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K-Cl function in EpoR-Cre;Kcc3f/f mice. We also report functional evidence suggesting incomplete recombinase-mediated excision of the Kcc3 gene in adult erythroid tissues. © 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.