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目的构建胃壁细胞定位表达SV40T的真核表达载体并制备转基因小鼠动物模型,为研究胃癌发病机制提供动物模型。方法从构建的胃壁细胞特异性表达载体pcDNA3.1(-)/HKSV中酶切回收3.8kb的基因片段H+-K+ATPaseβpromoter/SV40T,通过显微注射的方法制备转基因小鼠,PCR和Southern blotting检测阳性转基因小鼠并建系繁殖,RT-PCR检测基因的表达情况。结果将422枚注射过的受精卵移植给16只假孕雌鼠,共生出77只仔鼠,移植成功率为18.2%。在出生的77只仔鼠中,2#、4#、8#、16#、24#、51#、57#、61#、68#、73#经PCR检测为阳性首建鼠。除68#不育外,其他9个品系首建鼠共生出99只F1代鼠。8#品系23只F1代尚未发现阳性鼠,另8个品系F1代经PCR和Southern blotting检测发现31只阳性鼠,阳性率为40.8%(31/76)。RT-PCR检测F1代阳性鼠均仅在胃组织中有SV40T基因的表达,而在心、肝、肾、肺、食道、肠、骨骼肌等组织中均不表达。不育首建鼠处死解剖发现胃组织有肿瘤存在。结论建立了胃壁细胞定位表达SV40T基因的转基因小鼠动物模型。
Objective To construct eukaryotic expression vector of gastric adenocarcinoma cell line SV40T and prepare animal model of transgenic mice to provide an animal model for studying the pathogenesis of gastric cancer. Methods The 3.8kb H + -K + ATPaseβpromoter / SV40T gene fragment was isolated from the constructed gastric parietal cell specific expression vector pcDNA3.1 (-) / HKSV and the transgenic mice were prepared by microinjection. PCR and Southern blotting Positive transgenic mice were detected and propagated. RT-PCR was used to detect the gene expression. Results 422 injected fertilized eggs were transplanted to 16 females and 77 offspring were born. The success rate of transplantation was 18.2%. Among the 77 pups born, 2 #, 4 #, 8 #, 16 #, 24 #, 51 #, 57 #, 61 #, 68 # and 73 # were positive by PCR. In addition to the 68 # infertility, the other nine strains co-produced 99 F1 rats. Twenty-three F1 progenies of 8 # line had not found positive mice. The other 8 F1 lines detected 31 positive mice by PCR and Southern blotting, the positive rate was 40.8% (31/76). The F1 generation positive mice showed no expression of SV40T gene only in gastric tissues by RT-PCR, but not expressed in heart, liver, kidney, lung, esophagus, intestine, skeletal muscle and other tissues. Sterile first mouse sacrificed anatomical findings of gastric tumor presence. Conclusion The animal model of transgenic mice expressing gastric SV40T gene was established.