tTA/tetO-CCKR-2双转基因模型小鼠鉴定及生存时间分析  

Identification and survival analysis of tTA/tetO-CCKR-2 double transgenic mice

作　　者：高子清 阮思蓓 李丽 凌凤 唐晓琴 康清梅 罗斯译 罗静 唐亚平 唐明希 Gao Ziqing;Ruan Sibei;Li Li;Ling Feng;Tang Xiaoqin;Kang Qingmei;Luo Siyi;Luo Jing;Tang Yaping;Tang Mingxi(Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Pathology,Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Neurobiology,Southwest Medical University,Luzhou 646000,Sichuan Province,China;Guangzhou Women and Children’s Medical Center,Guangzhou 510000,Guangdong Province,China)

机构地区：[1]西南医科大学,四川省泸州市646000 [2]西南医科大学附属医院病理科,四川省泸州市646000 [3]西南医科大学神经生物教研室,四川省泸州市646000 [4]广州市妇女儿童医疗中心,广东省广州市510000

出　　处：《中国组织工程研究》2021年第11期1682-1687,共6页Chinese Journal of Tissue Engineering Research

基　　金：四川省科技厅科技支撑计划(14ZC0054),项目负责人:唐亚平;泸州市科知局-西南医科大学联合资助项目(2017LZXNYD-J14),项目负责人:唐明希。

摘　　要：背景:前脑特异性胆囊收缩素受体2(CCKR-2)双转基因小鼠(tTA/tetO-CCKR-2 tg,简称dtg)是焦虑相关疾病的理想动物模型,但尚缺乏该模型鉴定及寿命相关数据。目的:鉴定dtg小鼠子代DNA及胆囊收缩素受体2转基因前脑特异性表达并分析其生存情况。方法:由α-CaMKII/tTA单转基因小鼠和tetO-CCKR-2单转基因小鼠杂交,构建tTA/tetO-CCKR-2双转基因小鼠模型,取子代鼠尾提取DNA进行基因型鉴定;以野生型小鼠(WT小鼠)做对照,原位杂交特异性识别前脑胆囊收缩素受体2转基因表达;记录2年内dtg和WT小鼠(雌、雄分别各30只)的自然寿命进行生存分析。实验方案经西南医科大学实验动物伦理委员会批准;伦理审批号:20150068。结果与结论:①基因型鉴定:dtg实验小鼠预期基因片段大小与琼脂糖凝胶电泳结果相符;②原位杂交鉴定:dtg小鼠前脑区域呈现强烈的胆囊收缩素受体2杂交信号,而WT小鼠几乎未呈现;③寿命观察:雌、雄dtg小鼠中位生存时间分别为76周和77周,生存率均随小鼠的年龄增长而降低,且均分别显著低于雌、雄WT小鼠(均P<0.001);不同性别dtg小鼠间生存率差异无显著性意义(P=0.577);④结果表明,dtg小鼠可通过鼠尾提取DNA、PCR扩增、琼脂糖凝胶电泳鉴定基因型及原位杂交鉴定胆囊收缩素受体2转基因在前脑特异性表达;tTA/tetO-CCKR-2双转基因诱导可能会缩短小鼠的总体寿命,但对雌、雄dtg小鼠间的影响无显著差异。BACKGROUND:The inducible forebrain-specific cholecystokinin receptor-2(CCKR-2)double transgenic(tTA/tetO-CCKR-2 tg,abbreviated as dtg)mice are an ideal model of anxiety-related diseases.However,there is still a lack of model identification and life related data OBJECTIVE:To identify the genomic DNA of the offspring and the specific expression of CCKR-2 transgene in the forebrain,and to analyze the survival probability of dtg mice.METHODS:α-CaMKII/tTA single transgenic mice and tetO-CCKR-2 single transgenic mice were cross-fertilized to construct a dtg mouse model.The genomic DNA was extracted from the tail of the offspring,and the genotypes were detected by PCR and agarose gel electrophoresis.Wild-type(WT)mice were used as controls.In situ hybridization was used to detect the expression of CCKR-2.Survival of dtg mice and WT mice(30 females and 30 males)was observed and recorded within 2 years.The study protocol was approved by the Experimental Animal Ethics Committee of Southwest Medical University,with an approval No.20150068.RESULTS AND CONCLUSION:Agarose gel electrophoresis results showed the molecular weight of the PCR products of dtg mice was consistent with the expected target gene fragment.In situ hybridization results showed a strong signal of CCKR-2 was detected in the forebrain of dtg mice,but hardly present in the WT mice.The median survival time of dtg mice was 76 weeks in females and 77 weeks in males.The survival probability was decreased with age in dtg mice.The survival probability of WT mice was significantly better than that of dtg mice(P<0.001).There was no significant sex difference between males and females of dtg mice(P=0.577).Therefore,the specific expression of CCKR-2 transgene in the forebrain can be identified using PCR amplification,genomic DNA extraction,agarose gel electrophoresis,and in situ hybridization.tTA/tetO-CCKR-2 double transgenic induction may shorten the survival time of mice,but no significant difference is observed between the females and males of dtg mice.

关 键 词：双转基因小鼠 基因型鉴定 原位杂交 生存分析 焦虑症 恐惧行为 应激障碍 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]