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从遗传性高K、低氨基酸转运的犬外周血中分离单个核细胞，诱导出红血球祖细胞。首先将单个核细胞在含FCS的IMDM培养液和植物血凝素刺激的白细胞条件培养液中孵育，然后在含促红细胞生成素的IMDM培养液中培养。克隆了KCCI、水通道蛋白1(AQP1)和钠依赖谷氨酸转运体(GLAST)基因，克隆了KCC1和AQP1的全长cDNAs，并从红系祖细胞和肾脏细胞中克隆了GLAST基因片段。这些基因与已报道的其他动物的同源性很高。注射AQP1的CRNA的非洲爪哇卵母细胞比只注射水的卵母细胞渗透水多3倍以上。因此，AQP1在犬红细胞和肾脏细胞中起到了水的渗透作用。转KCC1基因的HEK293正在研究离子转运。未来，通过定点突变构建的突变基因将被导入细胞，以研究AQP1和KCC1在体积调节方面的合作。

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.