牙本质涎蛋白转基因小鼠的建立和转基因表达的初步分析

目的:构建牙本质涎蛋白(DSP)转基因小鼠,并对转基因表达进行初步RT-PCR分析。方法:将pcDNA3.1载体中的CMV启动子替换为启动子cβ-actin,构建载体pcDNA3.1-CX,然后将PCR获得的DSP基因编码序列克隆到pcDNA3.1-CX中,构建DSP转基因载体pcDNA3.1-CX-dsp;将线性化的载体DNA注射到小鼠受精卵的雄原核,将受精卵移植到假孕母鼠的输卵管。仔鼠出生后,用PCR及Southern印迹检测阳性小鼠,并用RT-PCR对其中一小鼠的F1代进行转基因表达分析。结果:共移植717枚注射过的受精卵至29只受体鼠,移卵后产仔67只,阳性4只。检测到53号小鼠F1代外源性DSP的表达。结论:通过显微注射方法,成功获得了DSP转基因小鼠,并证实外源性DSP可在53号小鼠F1代获得表达。

Generation of transgenic mouse of dentin sialoprotein and transgene expression analysis

PURPOSE: To generate the transgenic mouse model of DSP and perform transgene expression analysis by RT-PCR. METHODS: Plasmid pcDNA3.1-CX was constructed by substituting promoter cbeta-actin for CMV promoter of pcDNA3.1, and the ultimate transgenic vector, pcDNA3.1-CX-dsp, was constructed by cloning DSP coding sequence into pcDNA3.1-CX. The pcDNA3.1-CX-dsp plasmid was linearized and microinjected into the male pronucleus of the zygotes. The tail DNA of pups was tested by PCR and Southern blot. A member of F1 generation of one positive mouse was used to perform transgene expression analysis by RT-PCR. RESULTS: 717 embryos were implanted to 29 recipient pseudopregnant mice, 4 of the 67 pups carrying the transgene. Expression of DSP was detected in a member of F1 generation of one positive mouse by RT-PCR. CONCLUSION: Founders of the DSP transgenic mouse were obtained successfully, and the expression of DSP was primarily confirmed.