Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we ge...Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.展开更多
目的探讨斑马鱼肠道自发绿色荧光的原因及环孢素A(cyclosporine A,CsA)对自发荧光的影响,优化利用转基因(transgenic,Tg)斑马鱼Tg(abcb4:eGFP)筛选肿瘤多重耐药药物的方法。方法选取受精后5天(five day post fertilization,5 dpf)的野生...目的探讨斑马鱼肠道自发绿色荧光的原因及环孢素A(cyclosporine A,CsA)对自发荧光的影响,优化利用转基因(transgenic,Tg)斑马鱼Tg(abcb4:eGFP)筛选肿瘤多重耐药药物的方法。方法选取受精后5天(five day post fertilization,5 dpf)的野生型(wild type,WT)和Tg(abcb4:eGFP)斑马鱼幼鱼,观察其肠道自发荧光;将鲢鱼胆汁注射到WT斑马鱼胚胎后观察荧光情况;检测不同处理浓度和不同处理时间的CsA对WT斑马鱼肠道自发荧光的影响;10μmol/L CsA处理2 h对Tg(abcb4:eGFP)斑马鱼荧光检测的影响;Western blot检测CsA对Tg(abcb4:eGFP)斑马鱼b4型ATP结合盒(ATP-binding cassette b4,Abcb4)和增强型绿色荧光蛋白(enhanced green fluorescence protein,eGFP)表达的影响。结果斑马鱼肠道存在自发荧光,可影响Tg(abcb4:eGFP)斑马鱼的荧光检测准确度;鲢鱼胆汁在斑马鱼胚胎中可激发出绿色荧光;CsA可抑制斑马鱼肠道自发荧光,其中10μmol/L CsA处理2 h可达到最佳抑制效应,且不影响斑马鱼Abcb4和eGFP蛋白表达。结论斑马鱼肠道自发荧光可能为胆红素所致,10μmol/L CsA处理2 h可有效抑制斑马鱼肠道自发荧光,提高Tg(abcb4:eGFP)斑马鱼药物筛选时荧光检测的准确度。展开更多
基金supported by Chinese National Key Program on Basic Research (Nos. 2006CB943501 and 2006BAI23B01-3)Key Project for Drug Discovery and Development in China (No. 2009ZX09501-027)+1 种基金Key Project for Infectious Diseases in China (No. 2008ZX10002-016)E-Institutes of Shanghai Municipal Education Commission (E03003)
文摘Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.
