甘丙肽过表达转基因小鼠的制作和鉴定

Establishment and identification of GAL-overexpression transgenic mouse model

目的建立甘丙肽(galanin,GAL)过表达转基因小鼠,为后续研究甘丙肽基因功能提供动物模型。方法重组质粒PDGFβ-GAL经XbaⅠ、HindⅢ双酶切后,将连接有启动子的甘丙肽基因片段回收纯化,用显微注射法将甘丙肽基因注入400枚受精卵的雄原核中,选取发育正常的受精卵移植到假孕母鼠的输卵管壶腹部,待其产仔。用PCR方法鉴定子代小鼠基因型,蛋白印迹分析(Western blot)鉴定子代小鼠体内GAL蛋白表达情况。结果获得子代小鼠50只,有8只小鼠在基因组上整合有GAL基因,整合率为16%;Western blot显示转入的基因片段PDGFβ-GAL在脑组织中的表达水平比肝组织高。结论通过显微注射法成功建立了GAL过表达转基因小鼠模型。

Objective To construct the galanin-overexpression transgenic mouse model for investigating the function of galanin.Methods The PDGF β-GAL gene was amplified by restriction digestion of recombinante plasmid PDGF β-GAL with Xba Ⅰ and Hind Ⅲ.Secondly,the objective DNA fragment was microinjected into 400 male-pronucleus of fertilized ovums.Then the good fertilized ovums were transplanted into uterine tube ampulla of pseudocyesis female mouse.The genotype of transgenic line was identified by PCR and the expression level of the gene was determined by Western blot.Results There were 8 mice integrated in the genome from 50 offspring mice,and the integration rate was 16％.The expression level of PDGF β-GAL in brain tissues was higher than that in liver tissues by Western blot.Conclusion The galanin-overexpression transgenic mouse model could be successfully established by pronuclear microinjection.