MTA1转基因小鼠的构建及鉴定  

Establishment and identification of MTA1 transgenic mouse model

作　　者：薛洪省[1] 王海娟[2] 刘健[2] 张丽[3] 林晨[2] 詹启敏[2] 赵志龙[1] 钱海利[2] 

机构地区：[1]大连大学附属中山医院胸心外科,大连116001 [2]中国医学科学院肿瘤医院肿瘤研究所分子肿瘤学国家重点实验室,北京100021 [3]中国医学科学院实验动物研究所卫生部人类疾病比较医学重点实验室,北京100021

出　　处：《中国比较医学杂志》2013年第9期31-35,F0002,共6页Chinese Journal of Comparative Medicine

基　　金：国家自然基金项目(81071773、81101518);分子肿瘤学国家重点实验室自主课题(SKL-2013-12)

摘　　要：目的将人的MTA1外源基因整合到C57BL/6J小鼠中,构建稳定高表达MTA1的小鼠模型。方法通过RT-PCR方法克隆人的MTA1编码序列,将MTA1插入真核表达载体pcDNA3.1构建pcDNA3.1-MTA1载体,回收片段后利用显微注射技术将目的基因片段注入到受精卵的雄原核中,使用MTA1特异性的引物经PCR鉴定出基因型阳性的转基因小鼠,再利用Western-blot及免疫组化方法检测MTA1在转基因小鼠全身级织表达情况。结果成功构建了MTA1转基因注射片段。在320枚显微注射受精卵中挑选出300枚存活卵移植到10只ICR小鼠假孕受体的输卵管中,10只ICR小鼠均怀孕,移植成功率为100%,共生出子代鼠80只,经PCR检测其中共有9只整合了MTA1基因,整合率为11.25%。经PCR鉴定MTA1整合阳性的F1代小鼠,再经Western-blot和免疫组化分析检测MTA1表达水平在脑、肺、肝、肠等组织中表达明显增高,肾、骨骼肌表达无差异。结论成功构建了脑、肝、肺、结肠高表达MTA1的转基因小鼠,为进一步MTA1研究奠定了良好的研究模型。Objective To construct metastasis-associated tumor gene 1 （ MTA1 ） stably-over-expressing mouse model by inserting the full human CONA MTA1 CDS to the genome of C57BL/6J mouse. Method MTA1 gene was cloned from human CDNA with RT-PCR, The cloned MTA1 gene was then inserted into eukaryotic expression vector pcDNA3.1 to construct pcDNA3.1-MTA1 carrier. The transgenic mouse was produced by microinjection method and the genotype was detected by PCR with specific primers. The expression profiles of MTA1 in body tissues were illustrated by Western-blot and immunohistochemical method. Results Three hundred mouse zygotes were treated with transgenic vectors DNA by microinjection and then transplanted into ten artificially pregnant female mice. Ten of these mice were gravid. The transplantation rate was 100%. A total of 80 F0 mice were obtained, of which 9 were MAT1 COS-carriers by PCR screening. The positive rate of transgenic mice was 11.25% （9/80）. Four lines of MTA1 transgenic mouse were established. Distribution of MAT1 in trangeric mowse was tissue-spefic and the expression levels of the MTA1 gene in brain, liver, lung and colon from transgenic mouse were higher than those from control mice. But kidney and skeletal muscle showed no difference between transgenic and corotrol mice. Conclusion Transgenic mice stably overexpressing MATI in brain, liver, lung and cocon were established successfully and guarantee further MTA1 function exploration.

关 键 词：MTA1 转基因 构建 鉴定 

分 类 号：R332[医药卫生—人体生理学]