| 凌 凤,阮思蓓,唐晓琴,李 昕,高子清,李 丽,唐亚平,罗 静,唐明希.前脑特异性CCKR-2双转基因小鼠的繁育及基因鉴定[J].,2021,(20):3841-3844 |
| 前脑特异性CCKR-2双转基因小鼠的繁育及基因鉴定 |
| Breeding and Identification of CCKR-2 Double Transgenic Mice |
| 投稿时间：2020-11-27 修订日期：2020-12-23 |
| DOI：10.13241/j.cnki.pmb.2021.20.008 |
| 中文关键词: CCKR-2 转基因小鼠 聚合酶链反应 琼脂糖凝胶电泳 原位杂交 |
| 英文关键词: CCKR-2 Transgenic mice Polymerase chain reaction Agarose gel electrophoresis In situ hybridization |
| 基金项目:四川省科技厅科技支撑计划项目（14ZC0054）；西南医科大学-泸州市联合项目（2017LZXNYD-J14）；西南医科大学青年基金项目（2017-ZRQN-O74，2018-ZRQN-092)；西南医科大学附属医院项目（17153） |
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| 中文摘要: |
| 摘要 目的：繁殖及鉴定可调控前脑特异性胆囊收缩素受体2（Cholecystokinin receptor-2,CCKR-2）双转基因小鼠（tTA/tetO- CCKR-2 double transgenic，简称dtg），为进一步研究CCKR-2在焦虑相关疾病，如焦虑症、恐惧行为、创伤后应激障碍等发病过程中的作用及分子机制提供实验模型。方法：（1）利用α-CaMKII/tTA单转基因小鼠与tetO/CCKR-2单转基因小鼠杂交，所得子代尽可能远亲繁殖获得dtg小鼠，提取子代鼠尾基因组DNA，采用PCR法及琼脂糖凝胶电泳法鉴定PCR产物以确定其基因型；（2）采用原位杂交方法验证CCKR-2双转基因前脑特异性表达，筛选CCKR-2双转基因型及前脑特异性表达者作为继代种鼠和实验用鼠。结果：（1）PCR凝胶电泳图显示清晰的tTA（350 bp）和CCKR-2（550 bp）条带，野生型无条带显示，表明琼脂糖凝胶电泳结果与dtg模型预期基因片段大小相符；（2）原位杂交结果显示dtg小鼠前脑区域有强烈的CCKR-2表达而野生型小鼠不明显。此结果表明dtg双转基因小鼠在本实验室的成功建立与繁殖及继代，同时繁殖出更多的dtg小鼠。结论：通过正确的饲养繁育和基因鉴定方法能成功获得dtg双转基因小鼠，为本实验室进行后续相关研究奠定了基础。 |
| 英文摘要: |
| ABSTRACT Objective: To breed and identify inducible forebrain-specific cholecystokinin receptor-2(CCKR-2) transgenic (tTA/tetO-CCKR-2 tg, abbreviated as dtg) mice, in order to serve the dtg mice as models for further studying the molecular mechanism and pathogenesis of CCKR-2 in fear behavior, anxiety disorder, posttraumatic stress disorder morbidity and other related diseases. Methods: (1) The dtg mice were obtained by crossing α-CaMKII/tTA single transgenic mice with tetO/CCKR-2 single transgenic mice, and the offspring was obtained as distant relatives as possible. The genomic DNA was extracted from the tails tissue of the offspring mice. PCR and agarose gel electrophoresis were used to identify the PCR products. (2) In situ hybridization verified the specific expression of CCKR-2 double transgenic mice in the forebrain, and that dtg mice were selected as subculture and experimental mice. Results: (1) The clear bands of tTA (350 bp) and CCKR-2 (550 bp) were shown in the PCR gel electrophoresis pattern, while no band was shown in the wild type, which indicated that the agarose gel electrophoresis results were consistent with the expected target size of gene fragments in the dtg mice model. (2) In situ hybridization results showed that CCKR-2 was strongly expressed in the forebrain of dtg mice, but not in the wild type mice. The results showed that dtg double transgenic mice were successfully bred and identified in the laboratory, and more dtg mice were bred. Conclusion: The dtg double transgenic mice can be obtained successfully through correct breeding and gene identification, which provides references for the application of the model and lays a foundation for the follow-up studies in the laboratory. |
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