SV40T胃壁细胞定位表达转基因小鼠的建立  

ESTABLISHMENT OF TRANSGENIC MICE EXPRESSING SV40T IN PARIETAL CELLS

作　　者：陈辉[1] 金辉[2] 杜春艳[3] 顾鹏宇[4] 王纯耀[3] 张钦宪[2] 高翔[4] 

机构地区：[1]郑州大学基础医学院细胞生物学与医学遗传学教研室,郑州450052 [2]郑州大学基础医学院组织学与胚胎学教研室 [3]郑州大学实验动物中心,郑州450052 [4]南京大学模式动物研究所,南京210061

出　　处：《解剖学报》2008年第5期699-702,共4页Acta Anatomica Sinica

基　　金：河南省重大科技攻关计划资助项目(0623031400)

摘　　要：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 SV40 T antigen had been used widely in establishment of transgenic tumor animal models, which could induce cellular immortalization and carcinogenesis. The objective of the study was to construct the eukaryotic specific expression vector of SV40 large T antigen in gastric parietal cells and to establish transgenic mouse models with microinjection technique. Methods 3.gkb DNA fragment H^＋ -K^＋ ATPase β promoter/SV40T was released from specific expression vector of SV40 large T antigen in gastric parietal cells pcDNA3.1 （ - ）/HKSV and injected into oocytes to establish transgenic mice. Transgenic positive mice were detected by PCR and Southern blotting. Gene expression was measured by RT-PCR. Results 422 injected eggs were transferred to 16 pseudopregnant female mice and 77 offspring were bern, the frequency of transplant was 18.2%. In 77 live-bern mice, 2^#、4^#、8^#、16^#、24^#、51^#、57^#、61^#、68^#、73^#, were proved to be positive founder mice by PCR. 68^# founder was sterile, and 99 F1 mice were borne by the other nine founders. None F1 positive mouse was found in 8^# offspring, 31 F1 mice were proved to be positive by PCR and Southern blotting in the other eight pedigrees. The rate of positive mice was 40.8 % （31/76）. mRNA expression only existed in stomachs of positive transgenic mice, but not in their hearts, livers, kidneys, lungs, esophaguses, intestines, skeletal muscles etc. Gastric tumour had been detected in sterile founder mouse. Conclusion Transgenic mice expressing SV40T in parietal cells were useful models to gastric cancer researches.

关 键 词：SV40T抗原 SOUTHERN BLOTTING 定位表达 转基因小鼠 

分 类 号：R373.21[医药卫生—病原生物学] R734.2[医药卫生—基础医学]