MTA1转基因小鼠组织表达差异性的鉴定-中国试验动物学报.DOC

MTA1转基因小鼠的构建及鉴定 薛洪省1，2，王海娟1，刘健1，张丽3，林晨1，詹启敏1，赵志龙2，钱海利1 （1 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；2 大连大学附属中山医院 胸心外科，大连 116001；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） 【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基因，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连接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。 【关键词】 MTA1；转基因；构建；鉴定 Establishment and identification of MTA1 transgenic mouse model XUE Hong-sheng1,2, WANG Hai-juan1, LIU Jian1, ZHANG Li3, LIN Chen1, ZHAN Qi-min1, ZHAO Zhi-long2, QIAN Hai-li1 (1 State Key Laboratory of Molecular Oncology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Beijing 10002, China; 2 Department of Thoracic Surgery, Zhongshan Hospital, Dalian University, Dalian 116001, China; 3 Key Laboratory of Human Diseases Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Science, Beijing 10002, China.) 【Abstract】 Objective To construct metastasis-associated tumor gene 1(MTA1) stably-over-expressing mouse model by inserting the full human MTA1 CDS to the genome of C57BL/6J mouse. Method MTA1 gene was cloned form human with RT-PCR, The cloned MTA1 gene was then inserted eukaryotic expression vector pcDNA3.1 to construct of pcDNA3.1-MTA1 carrier. The transgenic mouse were 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 . Results Three hundred mouse zygotes were treated with transgenic vectors DNA by microinjection and then transplanted into ten artificial pregnant female mice. Ten of t