Brevican基因敲除小鼠的制备  

Generation of brevican knockout mice

作　　者：韩晞[1] 董艳[2] 

机构地区：[1]复旦大学附属华山医院神经外科,上海200040 [2]第二军医大学长征医院神经外科,上海200003

出　　处：《第二军医大学学报》2011年第8期818-821,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30571912)~~

摘　　要：目的制备brevican基因敲除小鼠。方法采用ET克隆方法,构建brevican打靶载体。线性化打靶载体,电转化胚胎干细胞(embryonic stem cell,ES),将正确同源重组的ES细胞注射入囊胚腔,生育嵌合体,再交配繁育杂合子及纯合子。PCR法鉴定小鼠的基因型。结果将PGK启动子指导的NEO表达框敲进Bcan第3外显子,敲除第3～8外显子序列,得到打靶载体,上游臂为2.4 kb,下游臂为4.8 kb。基因打靶后,得到双臂均发生正确同源重组的克隆数14个。利用阳性ES细胞克隆注射入囊胚,得到3只嵌合率大于50%的雄鼠,繁育得到6只Brevican-/-纯合子小鼠。结论利用同源重组方法,成功敲除干细胞靶基因,建立Brevican-/-建系小鼠。Objective To generate brevican knockout mutant mice.Methods The targeting construct to inactivate the brevican gene was made by using the ET cloning technology.The construct was linearized and electroporated into embryonic stem（ES） cells.Homologous recombinants were identifed by PCR after selecting the stable transfectants.The positive clones were then injected into C57BL/6 blastocysts,and the injected blastocysts were transferred into pseudopregnant foster mothers.Heterozygote and homozygote were multiplied by procreation mosaic.Progeny gene type was assayed by PCR.Results A neomycin resistance expression cassette under the control of the phosphoglycerate kinase（PGK） promoter was flanked by a 2.4-kb 5-arm fragment and a 4.8-kb 3-arm fragment,thus introducing the NEO gene into exon 3 and deleting the encoding region from exon 3 to exon 8 after homologous recombination.Fourteen clones of gene-targeted ES cells were identified and three male chimeras with a higher than 50% chimeric ratio were produced.Six brevican-null mice were generated after outbred and inbred.Conclusion Genetically modified ES cells have been successfully generated by homologous recombination,and brevican-deficient mouse strain has been successfully generated.

关 键 词：BREVICAN 基因敲除小鼠 胚胎干细胞 同源重组 

分 类 号：R-332[医药卫生]