海马与新皮质组织特异性<i>GABRG2</i>基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 <sup>*</sup>

doi:10.13523/j.cb.1908034

中国生物工程杂志

2020, Vol. 40

Issue (3): 9-20 DOI: 10.13523/j.cb.1908034

研究报告

海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 ^*

郭胜楠^1,^2,^**,李信晓^2,^3,^**,王峰^2,³,刘昆梅²,丁娜²,扈启宽^1,^2,^***(

),孙涛^2,^3,^***(

)

1 宁夏医科大学基础医学院银川 750004
2 宁夏医科大学宁夏颅脑疾病重点实验室银川 750004
3 宁夏医科大学总医院银川 750004

Establishment and Identification of the Neocortex and Hippocampus GABRG2 Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus

GUO Sheng-nan^1,^2,^**,LI Xin-xiao^2,^3,^**,WANG Feng^2,³,LIU Kun-mei²,DING Na²,HU Qi-kuan^1,^2,^***(

),SUN Tao^2,^3,^***(

)

1 Department of Physiology, Basic Medical School of Ningxia Medical University, Yinchuan 750004, China
2 Ningxia Key Laboratory of Cerebrocranial Diseases, Yinchuan 750004, China
3 Department of Neurosurgery, General hospital of Ningxia Medical University, Yinchuan 750004, China

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摘要：

建立基于Cre/loxp重组酶系统调控的海马和新皮质特异性GABA_A受体γ2亚基(GABRG2)基因条件基因敲除小鼠模型,为深入研究海马区和新皮质GABRG2在癫痫发生中的功能作用提供动物模型.将引进的GABRG2 ^fl/wt转基因小鼠与海马和新皮质特异性表达Cre ^+/+重组酶工具鼠分别进行繁配和鉴定,然后再将2种小鼠进行杂交并对其子代小鼠的基因型进行鉴定,其子代基因型为GABRG2 ^fl/wtCre ⁺的小鼠为构建的海马区和新皮质特异性GABRG2基因条件性敲除小鼠.利用PCR技术鉴定小鼠基因型,Real-Time PCR和Western blot技术检测GABRG2基因在小鼠海马和新皮质中的mRNA水平和蛋白质水平的表达情况.PCR结果显示子代小鼠基因型符合GABRG2 ^fl/wtCre ⁺;海马与新皮质特异性GABRG2基因敲除小鼠海马和新皮质中GABRG2的mRNA水平和蛋白质水平显著低于对照组;热造模过程中,实验组小鼠癫痫发作更明显.利用Cre/Loxp技术成功构建了海马与新皮质GABRG2基因敲除小鼠,可为进一步研究GABRG2在癫痫发生中的作用机制奠定基础.

关键词： GABRG2基因; 遗传性癫痫伴热性惊厥附加症; 海马与新皮质; Cre-Loxp重组酶系统基因敲除

Abstract:

To establish and identify the neocortex and hippocampus specific GABA_A receptor γ2 subunit (GABRG2) knockout mice via Cre/Loxp conditional gene knockout technology, and provide the important animal model for further investigating the functional role of GABRG2 in hippocampus and neocortex GABRG2 in epileptogenesis. The constructed GABRG2 ^fl/wt mice and the mice that express Cre recombinase to approximately 88% of the neurons of the neocortex and hippocampus were bred and authenticated, respectively. Mating and identification of GABRG2 ^fl/fl mice with Cre mice were carried out, and the GABRG2 ^fl/wt Cre ⁺ mice were screened. The mouse genotypes were identified by PCR. The Real-Time PCR and Western blot were used to detect the expression of GABRG2 mRNA and protein levels in mouse neocortex and hippocampus. PCR results indicate that mouse genotypes are consistent with GABRG2 ^fl/wt Cre ⁺. Compared with control group mice, GABRG2 mRNA and protein levels were significantly reduced. During the temperature elevation, the GABRG2 ^fl/wt Cre ⁺ mice had significant seizures. Based on the Cre/loxp conditional gene knockout technology, that succeed in building a group of neocortex and hippocampus specific GABRG2 gene knockout mice which can stably go down to the future generation, which would supply a technical basis for animal models in further researches of the regulation and mechanism of GABRG2 gene in the progress of epilepsy.

Key words: GABRG2 Genetic epilepsy with ebrile eizures plus Neocortex and hippocampus Cre/Loxp recombination system Gene knockout

收稿日期: 2019-08-20 出版日期: 2020-04-18

ZTFLH:

Q78

基金资助: * 宁夏回族自治区"十三五"重大科技项目(2016BZ07);国家自然科学基金(81660226,81971085)

通讯作者: 扈启宽,孙涛 E-mail: huqikuan@163.com;suntao6699@163.com

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引用本文:

郭胜楠, 李信晓, 王峰, 刘昆梅, 丁娜, 扈启宽, 孙涛. 海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(3): 9-20.

GUO Sheng-nan, LI Xin-xiao, WANG Feng, LIU Kun-mei, DING Na, HU Qi-kuan, SUN Tao. Establishment and Identification of the Neocortex and Hippocampus GABRG2 Knockout Mice and Its Preliminary Study in Generalized Epilepsy with Febrile Seizures Plus. China Biotechnology, 2020, 40(3): 9-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1908034 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I3/9

图1 海马和新皮质组织特异性GABRG2基因敲除小鼠构建策略

图2 GABRG2 flox小鼠鉴定引物设计示意图

表1 GABRG2 flox小鼠基因型鉴定的引物信息

表2 GABRG2fl/wtCre+小鼠基因型鉴定的引物信息

图3 F0代与背景鼠回交子代基因型PCR鉴定结果

图4 表达Cre重组酶的小鼠基因型鉴定结果

图5 基因型GABRG2fl/wtCre+/-小鼠与GABRG2fl/wtCre+/-小鼠繁配后代基因型鉴定结果

图6 GABRG2蛋白在NC组和KO组的表达水平

图7 小鼠颅内脑电图电极植入

图8 升高体内核心温度诱发GABRG2fl/wtCre+小鼠癫痫发作,而对照组小鼠无癫痫发作

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