摘要
采用遗传学育种方法 ,使外源基因整合位点随机的基质金属蛋白酶抑制剂 1(TIMP 1)转基因小鼠成为单一整合位点的纯合子转基因小鼠而建立TIMP 1转基因小鼠品系 .通过受精卵原核显微注射方法 ,获得带有人TIMP 1基因的Founder小鼠 .将转基因小鼠与正常小鼠交配 ,得到子代小鼠 .通过PCR及Southern印迹等方法 ,检测TIMP 1DNA在转基因小鼠体内的整合情况 ,阳性率达5 0 %后 ,进行近亲交配 .提取小鼠组织总RNA ,Northern印迹分析阳性小鼠各组织外源性TIMP 1mRNA表达情况 ,以正常NIH小鼠做对照 .获得了 6代小鼠共 4 2 4只 ,其中PCR阳性鼠 2 72只 ,Southern阳性鼠 2 2 6只 ,纯合子转基因小鼠 12 8只 ;F4代后阳性率达到 95 %以上 .转基因小鼠TIMP 1基因表达情况在肾脏的丰度明显高于肝脏和脾脏 (P <0 0 1) ,而肝和脾之间并没有显著差异 (P>0 0 5 ) .外源基因在转基因小鼠体内可以稳定遗传 ,并得到了整合有TIMP 1基因的纯合子转基因小鼠 ,且在阳性的转基因小鼠体内在肾脏中特异性表达 ,为以后开展TIMP
The homozygote of human tissue inhibitor of metalloproteinase 1(TIMP 1) transgenic mice with unique integrated site was obtained by genetic breeding. To generate Founder transgenic mice, the recombinant plasmids harboring the human TIMP 1 gene were introduced into the male pronuclei of mouse fertilizer eggs by microinjection. The offsprings generated from the Founders were mated with other NIH mice. The integration ratios of target gene were tested by polymerase chain reaction (PCR) and Southern blotting. When the integration ratio was almost 50%, the positive mice were mated in inbreeding. The TIMP 1 mRNA expression in some tissues was analyzed by using Nothern blotting. The six generations of human TIMP 1 transgenic mice (128 homozygote) were obtained. After F 4 the integration ratio was higher than 95%. The expression of human TIMP 1 mRNA of the transgenic mice was significantly higher in kidney than in liver and spleen ( P <0 05), but it was no significant difference between the liver and spleen, and there was no expression in the brain. The human TIMP 1 gene can be stably transmitted in the transgenic mice, and the homozygote of human TIMP 1 transgenic mice was obtained. The gene was specially expressed in the kidney. The transgenic mice would be helpful for the study of the nephro pathophysiology.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第5期558-562,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目 (No .3 0 0 0 0 0 80 )
国家自然科学基金"创新研究群体"基金项目(No .3 0 12 2 10 0 5 )
国家"973"项目(No.G2 0 0 0 0 5 70 0 3)资助~~