Dog red blood cells possessing hereditary high K and low amino acid transport as variant model (s) for abnormal differentiation of erythroid progenitor

用Ficoll-Conray法从遗传性高钾低氨基酸转运犬外周血中分离单核细胞诱导红细胞祖细胞。先在含FCS的IMDM培养基和植物血凝素刺激的白细胞条件培养基中培养单核细胞，再在含促红细胞生成素的IMDM培养基中培养。这些细胞含有联苯胺、糖蛋白和血红蛋白，因此证实这些细胞已分化为红细胞。对K-Cl共转运蛋白（KCCI）、水通道蛋白1 （AQP1）和na依赖性谷氨酸转运蛋白（GLAST）进行cDNA克隆，克隆了KCC1和AQP1的全长cDNA，并从红系祖细胞和肾细胞中克隆了GLAST片段cDNA。这些dna与报道的其他动物的dna高度相同。注射AQP1的cRNA的爪蟾卵母细胞比只注射水的卵母细胞渗透性高3倍以上。因此，我们强烈建议AQP1参与狗红细胞和肾细胞的水渗透。转染KCC1的HEK293正在研究离子转运。未来，我们将通过位点定向诱变构建突变基因，转染到细胞中，研究AQP1和KCC1在体积调控中的协同作用。

Progenitor of red blood cells were induced from mononuclear cells separated by Ficoll-Conray from peripheral blood in dog with hereditary high K and low amino acid transport. Firstly the mononuclear cells were incubated in IMDM medium with FCS and phytohemaglutinin-stimulated leukocyte conditioned medium, then they were cultured in the IMDM medium with erythropoietin. These cells were positive with benzidine, glycophorin and hemoglobin, thus it was confirmed that these cells differentiated to erythroblasts.cDNA cloning of K-Cl cotransporter (KCCI) , aquapolin 1 (AQP1) and Na-dependent glutamate transporter (GLAST) was performed, and full length cDNAs of KCC1 and AQP1 were cloned, and fragment cDNA of GLAST was cloned from the erythroid progenitor and kidney cells. These cDNAs were highly identical to those of other animals reported.Xenopus oocytes injected with cRNA of AQP1 permeated water more than 3-fold compare to the oocytes injected with only water. Therefore, it was strongly suggested that the AQP1 act for water permeation in the dog red blood cells and kidney cells. HEK293 transfected with KCC1 are investigating for ion transport. In future, mutant gene constructed by site directed mutagenesis will be transfected to the cells to investigate cooperation of AQP1 and KCC1 for volume regulation.