胰岛B细胞特异性敲除基因APPL1小鼠模型的制备  

Establishment of pancreatic islet beta cell-specific APPL1 knockout mouse model

作　　者：李晓雯[1] 李羚[1] 林紫薇[1] 孙赟[1] 陈辰 王琛[1] 贾伟平[1] 

机构地区：[1]上海交通大学附属第六人民医院内分泌代谢科,上海市糖尿病研究所,上海市糖尿病重点实验室,上海200233 [2]上海南方模式生物研究中心

出　　处：《上海医学》2015年第3期231-234,共4页Shanghai Medical Journal

基　　金："十一五"国家科技支撑计划重点项目(2006BAI23B02);上海交通大学医学院-渥太华大学联合医学研究项目资助

摘　　要：目的制备胰岛B细胞特异性APPL1(adaptor protein containing PH domain,PTB domain and Leucine zipper motif 1)基因敲除小鼠模型(B-APPL1KO)。方法采用基因剔除打靶技术,胚胎干细胞(ES)基因打靶,囊胚显微注射法制备嵌合小鼠,嵌合小鼠与野生型C57BL/6J雌性小鼠繁殖APPL1-flox杂合子(APPL1flox/+)小鼠,APPL1-flox纯合子(APPL1flox/flox)小鼠由APPL1flox/+小鼠相互交配获得。利用Cre-LoxP系统繁育B-APPL1KO小鼠。繁育小鼠经尾端裂解提取DNA鉴定基因型,采用Western印迹法检测APPL1蛋白表达水平。结果 APPL1flox/flox和B-APPL1KO小鼠繁育成功。APPL1flox/flox小鼠胰岛组织有APPL1表达,而B-APPL1KO小鼠则无APPL1表达。APPL1flox/flox和B-APPL1KO小鼠的脂肪组织均有APPL1蛋白表达。与APPL1flox/flox小鼠相比,B-APPL1KO小鼠的体重有增加的趋势,但两者间各时间点的差异均无统计学意义(P值均>0.05)。两者间8、10周龄的空腹血糖和10周龄的空腹胰岛素水平的差异均无统计学意义(P值均>0.05)。结论胰岛B细胞特异性敲除基因APPL1小鼠模型制备成功,为探讨APPL1在胰岛中的功能提供研究工具。Objective To establish APPL1 （adaptor protein containing PH domain, PTB domain and Leucine zipper motif 1 in pancreatic islet beta cells） knock out mouse model （B-APPL1Ko） in B cells. Methods Mouse embryonic stem （ES） cells were targeted by knockout targeting technology, and blastula microinjection was used to obtain chimera mouse. Then chimeric mice breeded with wild type C57BL/6J mice to obtain APPLI-flox heterozygous mice （APPL1^flox＋ ）. APPLI-flox homozygous mice （APPL1^flox/flox） were generated by cross-breeding APPL1^flox＋ mice. Cre-loxp system was used to obtain B-APPL1^Komice. Genetype of mice was determined by tail DNA cracking and extraction. Protein expression of APPL1 was measured by Western blotting. Results APPL1^flox/floxand B-APPL1^komice were successfully generated. APPL1 protein was found in pancreatic islet of APPL1^flox/flox mice, but not in B-APPL1^ko mouse islets. However, APPL1 protein was found in fat tissue of both APPL1^flox/flox and B-APPL1^ko mice. B-APPL1^ko mice had decreased weight than APPL1^flox/flox＇ ones; however, no statistical significance was found between them （P 〉0.05）. There were no significant differences in terms of fasting plasma glucose （8 or 10 weeks old） or fasting insulin levels （10 weeks old）between APPL1^flox/flox and B- APPL1^komice （all P 〉 0. 05）. Conclusion The conditional APPLl-knockout mouse model is successfully established. It lays a foundation for study on APPL1 in mouse islets.

关 键 词：APPL1 胰岛B细胞 条件性敲除 

分 类 号：R587.1[医药卫生—内分泌]