海马神经细胞特异性DRD2基因条件性敲除小鼠模型的建立与鉴定  

Establish ion and Identification of Hippocampus Nerve Cells Specific DRD2-conditional Knock-out Mice Models

作　　者：段朝霞[1] 陈魁君[1] 张东冬 张洁元[1] 王建民[1] DUAN Zhao-xia;CHEN Kui-jun;ZHANG Dong-dong;ZHANG Jie-yuan;WANG Jian-min(The Sixth Department of Research Institute of Battle Surgery, Daping Hospital of PLA Army Military Medical University, Daping, Chongqing 400042, China)

机构地区：[1]陆军军医大学大坪医院野战外科研究所第六研究室,重庆400042

出　　处：《解放军医药杂志》2019年第4期1-5,共5页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：国家自然科学基金资助项目(81471860)

摘　　要：目的拟构建DRD2基因大脑海马组织特异性敲除小鼠模型,旨在进一步深入研究DRD2的生物学功能。方法运用逆转录病毒基因标记技术,结合Cre-loxP重组酶系统,设计和构建打靶载体,然后将打靶载体电转至小鼠胚胎细胞,用PCR和Southern blot验证ES克隆的正确性,最后将筛选出的ES显微注射到胚囊内,经过PCR鉴定得到Neo delete杂合子小鼠(flox/+)。将Neo delete杂合子小鼠(flox/+)自交获得Neo delete纯合子小鼠(flox/flox)小鼠,经PCR筛选出的在DRD2^(flox/flox)纯合子小鼠与海马组织特异性表达Cre基因的Ca MKⅡα-Cre工具鼠杂交,获得了DRD2^(flox/flox)的特异性组织条件性敲除小鼠,并用定量PCR和Western blot法对其进行鉴定。利用PCR和Western blot方法检测成年DRD2条件性敲除小鼠大脑不同部位(前额、海马)的DRD2表达情况。结果经过多层筛选、鉴定,最后得到DRD2^(flox/flox)的特异性组织条件性敲除小鼠。所制备的DRD2条件性敲除小鼠基因缺失主要发生在海马组织中,前额组织中表达水平变化不大。DRD2表达水平在不同小鼠的海马组织中降低。DRD2海马组织特异性敲除小鼠在交配饲养过程中纯合子小鼠未出现胚胎致死现象,也未发现其他器官组织结构的异常改变。结论成功建立大脑海马神经细胞特异性DRD2基因敲除模型,为进一步深入研究DRD2的生物学功能,尤其是DRD2在精神类疾病的发生、发展中的作用机制提供了研究基础。Objective To further study DRD2biological function by establishing mice models of hippocampus never cells specific DRD2-conditional knock-out mice. Methods Targeting vectors were designed and constructed by using retroviral gene marker technology and Cre-loxP recombinase system, and then target vectors were electronically transferred to mouse embryonic cells. Correctness of ES cloning was verified by polymerase chain reaction (PCR) and Southern blot. Finally, selected ES was microinjected into embryo sac. Neo delete heterozygote mice (flox/+) were obtained which was identified by PCR. Neo delete homozygous mice (flox/flox) were obtained by self-crossing Neo delete heterozygous mice (flox/+). DRD2 flox/flox homozygous mice, which had selected by PCR, were hybridized with CaMKIIα-Cre tool mice expressing Cre gene specifically in hippocampus tissues, and then specific tissue conditional knockout mice of DRD2 flox/flox were obtained and identified by quantitative PCR and Western blot methods. DRD2 expressions in different parts of the brain (forehead and hippocampus) of adult DRD2 conditioned knockout mice were detected by PCR and Western blot methods. Results DRD2 flox/flox specific tissue conditioned knockout mice were finally obtained after multi-layer screening and identification. Gene deletion of the prepared CDRD2 conditional knockout mice mainly occurred in hippocampus tissues, while the expression in forehead tissue showed little change. DRD2 expression was decreased in hippocampus tissues of different mice. During mating and feeding, DRD2 hippocampal tissue-specific knockout mice did not have embryolethality or abnormal changes in structures of other organs. Conclusion Successful establishment of a specific DRD2 gene knockout model in cerebrum hippocampal neurons provides basis for further study of DRD2 biological functions, especially DRD2 mechanism in occurrence and development of psychiatric diseases.

关 键 词：DRD2 小鼠 基因敲除 精神病 

分 类 号：R-332[医药卫生] R74