Immunological analyses and gene therapy in chronic colitis of IL-12p40 transgenic mice

目的：为探讨IL-12p40在慢性肠炎中的作用，我们利用T_3b启动子构建了IL-12p40转基因小鼠，并检测了IL-12p40对肠上皮细胞的限制性表达。(方法与结果)：用DNA显微注射法建立T3、b-IL-12p40转基因小鼠。转基因包括T_3^b启动子、兔B珠蛋白外显子和内含子、小鼠IL-12p40基因和兔β珠蛋白A基因。他们的表型几乎正常。将这些小鼠与C57BL/6小鼠回交，并在4个后代中获得后代，系#9、#13、#20和#24。Southern印迹法检测小鼠基因组中反式基因的拷贝数。从条带杂交强度判断，4只转基因小鼠携带的T_3^b-IL-12p40不到10个拷贝。Northern印迹分析和RT-PCR检测Trangene来源的mRNA的表达。在转基因小鼠的大肠和小肠中均检测到IL-12p40基因的表达。除胃肠道外，在包括胸腺在内的主要器官中未检测到转基因产物。用IL-12(p40/p70)单抗对转基因小鼠和阴性仔鼠的大肠组织进行免疫组织化学分析。转基因小鼠大肠上皮细胞主要染色。用1.5%葡聚糖硫酸钠诱导T3、b-IL-12p40转基因小鼠结肠炎和阴性仔鼠。转基因小鼠与阴性仔鼠的结肠炎评分差异不显著。

(Aims) : To investigate the roles of IL-12p40 in chronic intestinal inflammation, we generated IL-12p40 transgenic mice using T3^b promoter and determined the restricted expression of IL-12p40 to intestinal epithelial cells. (Methods & Results) : T3^b-IL-12p40 trans genic mice were produced by DNA microinjection method. Transgene contains T3^b promoter, rabbit B globin exon and intron, mouse IL-12p40 cDNA and rabbit beta globin polyA.As a result of screenings by PCR, six founders carrying this transgene were produced. Their phenotypes were almost normal. These mice were backcrossed with C57BL/6 mice, and next generations were obtained in four founders, line #9, #13, #20 and #24. The copy number of the trans gene in mouse genome was determined by Southern blot analysis. As judged from hybridizing intensity of bands, the four transgenic mice carry less than 10 copies of the T3^b-IL-12p40 construct. Northern blot analysis and RT-PCR were performed to examine mRNA expression derived from trangene. IL-12p40 mRNA was detected in large and small intestine of the transgenic mice. Except for gastrointestinal tract, mRNA derived from transgene was not detected in major organs including thymus. And immunohistochemical analysis of the large intestine derived from transgenic mouse and negative littermate were conducted using IL-12 (p40/p70) monoclonal antibody. The epithelial cells of the large intestine in transgenic mice was mainly stained. Colitis was induced in T3^b-IL-12p40 transgenic mice and negative littermate by 1.5% dextran sulfate sodium. The differences of colitis score between transgenic mice and negative littermate were not significant.