Tet-on系统诱导乳腺特异表达CUEDC2转基因小鼠的制备

摘要

A CUEDC2 transgenic expression vector was constructed first.After expression test in MCF7-rtTA cells, the vector was digested by restriction enzymes.The DNA fragments were microinjected into fertilized eggs of mice.Then the manipulated embryos were transferred into the oviducts of pseudopregnant recipient female mice, and the pups of 3 founders were identified by PCR analysis.Next, CUEDC2 transgenic mice were crossed with the MMTV-rtTA transgenic mice for CUEDC2/rtTA double transgenic mice.The CUEDC2 expression of the offsprings were analyzed by PCR, RT-PCR and Western blot assays.Twelve mice were identified as carrying copies of transgene.Immunohistochemical analysis and Western blotting results showed that the transgene of CUEDC2 was specific expressed in mammary gland in 2 transgenic mouse lineages.In a word,we established a CUEDC2/rtTA double transgenic mouse model, which will facilitate the investigation of the biological function of CUEDC2 in vivo.%建立Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠模型,构建转基因表达载体,经细胞诱导表达验证后,酶切得到含有CUEDC2的线性DNA片段,并采用显微注射的方法及后续筛选鉴定,得到CUEDC2转基因小鼠.进而通过与乳腺特异表达的MMTV-rtTA转基因小鼠交配,得到CUEDC2/rtTA双阳性转基因小鼠.利用2 mg/mL的多西环素(Doxycycline)诱导小鼠体内CUEDC2表达.通过Western Blot、免疫组化检测表达.结果表明经诱导CUEDC2/rtTA双阳性转墓因小鼠的2个Founder的F1、F2代在转录水平和蛋白水平均成功表达CUEDC2,并具有乳腺特异性.表明Tet-on系统诱导乳腺特异表达CUEDC2的转基因小鼠制备成功.为后续CUEDC2的功能研究奠定了技术基础.