成年大脑神经细胞特异性ADAM10基因敲除小鼠模型的建立与鉴定

Generation and characterization of the adult neuron-specific ADAM10 knock-out mice

去整合素和金属蛋白酶10(ADAM10)是一种能够水解30余种跨膜蛋白质的"脱落酶"(sheddase),参与诸多生理过程和致病机制,如胚胎发育、细胞粘附、信号转导、免疫反应、癌症和阿尔茨海默病。迄今,已报道的ADAMIO完全基因敲除小鼠和大脑神经前体细胞特异性ADAM10基因敲除小鼠分别于胚胎期或围产期死亡,致使无法研究成年小鼠大脑神经细胞ADAM10基因的功能。文章利用本研究小组建立的CaMKlla-Cre转基因小鼠与ADAM10loxP/loxP转基因小鼠杂交,获得了CaMKIIα-Cre/ADAM10loxP/loxP小鼠,并对其进行鉴定。利用PCR方法检测成年ADAM10cKO小鼠大脑基因组DNA表明,ADAM10基因缺失主要发生在前脑皮层和海马中。荧光定量PCR检测结果显示,ADAM10 mRNA的表达水平在前脑皮层和海马中分别降低55.7%和60.8%;使用Western blotting方法研究发现,ADAM10成熟蛋白质的含量在前脑皮层和海马中分别减少63%和84.8%。采用免疫组织化学方法检测表明,成年ADAM10cKO小鼠与野生型小鼠相比,其大脑皮层和海马神经细胞的ADAM10免疫染色明显减弱,而其它细胞如胶质细胞的免疫染色基本一致。总之,文章成功制备了首个存活至成年的大脑神经细胞特异性ADAM10基因敲除(cKO)小鼠,克服了小鼠因ADAM10缺失在胚胎期或围产期死亡的弊端,为研究成年小鼠大脑神经细胞ADAMIO基因的功能奠定了坚实的基础。

A disintegrin and metalloproteinase 10（ADAM10） is a major sheddase for over 30 different membrane proteins and gets involved in such physiological processes and pathogenesis as embryonic development,cell adhesion,signal transduction,immune reaction,cancer,and Alzheimer＇s disease.Both ADAM10 knock-out mice and the neural progenitor cell-specific ADAM10 knock-out mice having been reported so far died in the embryonic or perinatal stage,respectively,thus resulting in the failure to investigate ADAM10 function in the adult mouse brain.Through a series of tests,we have succeeded in generating and characterizing the CaMKIIa-Cre/ADAM10loxP/loxP mice surviving until adulthood by means of crossing ADAM10loxP/loxP mice with newly generated CaMKIIa-Cre transgenic mice.PCR analysis of genomic DNAs from different regions of the ADAM10 cKO mouse brain shows that the deleted ADAM10 alleles are mainly found in the cortex and hippocampus.Real-time RT-PCR findings further confirm that ADAM10 mRNAs decrease in the cortex and hippocampus by 55.7% and 60.8%,respectively.Western-blotting analysis demonstrates 63% and 84.8% loss of mature ADAM10 proteins from the cortex and hippocampus.Immunohistochemical tests show that there is significantly less ADAM 10positive staining in the cortical and hippocampal neurons but not gliocytes of ADAM10 cKO mice compared with control mice.In summary,we established the adult neuron-specific ADAM10 knock-out（cKO） mice for the first time,which prevented ADAM10-/-mice from the embryonic and perinatal mortality and laid a firm foundation for the further study of ADAM10 function in the brain of adult mice in vivo.