摘要
目的:应用Cre-Loxp系统构建Claudin-7蛋白在肠道可诱导性条件性基因敲除小鼠模型,并进行表型分析.方法:构建Claudin-7打靶载体,通过电击法转染入小鼠胚胎干细胞(ES细胞),用正负筛选法筛选阳性ES细胞并进行PCR鉴定和Southern鉴定,利用显微注射把发生正确同源重组的ES细胞注射进C57BL/6N小鼠囊胚,移入受体小鼠子宫以获得嵌合体小鼠.将得到的嵌合体小鼠与C57BL/6N小鼠交配获得Claudin-7-flox小鼠,该小鼠与PvillinCre ERT2转基因小鼠杂交,通过子代自交获得Claudin-7在肠道可诱导性条件性基因敲除(Cldn7fl/fl Villin-Cre ERT2)的小鼠,他莫昔芬(tamoxifen)药物诱导肠道Claudin-7基因敲除,对小鼠进行表型分析.结果:获得Claudin-7-flox小鼠及Claudin-7肠道可诱导性条件性基因敲除小鼠数只,Claudin-7肠道可诱导性条件性基因敲除小鼠出生时表型正常,发育良好,他莫昔芬(tamoxifen)药物应用后,诱导了小鼠肠道上皮细胞Claudin-7基因表达的缺失.小鼠出现脱水表现,活泼度降低,并出现死亡.结论:成功构建了Claudin-7肠道可诱导性条件性基因敲除小鼠模型,利用他莫昔芬诱导肠道Claudin-7基因敲除,初步构建了肠道炎症模型,为进一步研究Claudin-7在肠道肿瘤中的作用奠定了基础.
AIM：To create an inducible Claudin-7conditional knockout（ICKO） mouse line using the Cre/ LoxP system and analyze the phenotype after inducing knockout in the intestine.METHODS：Claudin-7 conditional knockout targeted vector was constructed and then transfected into embryonic stem（ES） cells by electroporation.The targeted ES cells were screened by positive-negative selection,and identified by PCR and Southern blot.The screened targeted ES cells were micro injected into the blastula of C57BL/ 6N mice.Chimerical mice were generated after transplanting the blastula into the host mice,and then the Claudin-7-floxed mice were generated by crossing between the chimerical mice and C57BL/6N mice.Claudin-7 ICKO mice could be obtained by crossing between PvillinCreERT2 transgenic mice and Claudin-7-floxed mice.A further phenotype analysis was made by HE staining.RESULTS：We obtained Claudin-7-floxed mice and Cldn7fl/ flVillin-CreERT2 homozygous mice successfully.The deletion of Claudin-7gene expression in mouse intestinal epithelial cells was successfully induced by tamoxifen.After tamoxifen induction,the mice developed dehydration,the degree of living activity decreased,and death occurred.CONCLUSION：We have successfully created an inducible Claudin-7 conditional knockout mouse line and induced the gene deletion in the intestine using tamoxifen,which may be used as a model of intestinal inflammation.
出处
《世界华人消化杂志》
CAS
2015年第31期5017-5023,共7页
World Chinese Journal of Digestology
基金
国家自然科学基金面上资助项目
No.81372585
北京市教育委员会科技计划基金面上资助项目
No.KM201410025026
2014年北京市留学人员科技活动择优资助优秀类项目~~
