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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 prepare brevican knockout mice. Methods The cloning method was used to construct brevican targeting vector. The target vector was linearized and transformed into embryonic stem cells (ES). The ES cells with the correct homologous recombination were injected into the blastula cavity to produce chimeras, and then mating and breeding of heterozygotes and homozygotes. The genotypes of mice were identified by PCR. Results The NEO expression vector guided by PGK promoter was knocked into exon 3 of Bcan, and the exons 3 ~ 8 were knocked out. The targeting vector was 2.4 kb upstream and 4.8 kb downstream. After gene targeting, 14 clones with correct homologous recombination in both arms were obtained. Positive ES cell clones were injected into the blastocysts to obtain 3 male mice with a chimeric rate greater than 50%, and 6 Brevican - / - homozygous mice were bred. Conclusion The homologous recombination method was used to successfully knock out the stem cell target genes and establish Brevican - / - lineage mice.