MTA1转基因小鼠的构建及鉴定

目的将人的MTAl外源基因整合到C57BL／6J小鼠中，构建稳定高表达MTAl的小鼠模型。方法通过RT—PCR方法克隆人的MTAl编码序列，将MTAl插入真核表达载体pcDNA3．1构建pcDNA3．1-MTAl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MTAl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MTAl在转基因小鼠全身级织表达情况。结果成功构建了MTAl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MTAl基因，整合率为11．25％。经PCR鉴定MTAl整合阳性的F1代小鼠，再经Western—blot和免疫组化分析检测MTAl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MTAl的转基因小鼠，为进一步MTAl研究奠定了良好的研究模型。

Establishment and identification of MTA1 transgenic mouse model

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.