海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。

Construction of Hippocampal Cortical Specific Knockout AEG-1 Gene Mice and Preliminary Study on Its Behavior

Astrocyte upregulating gene-1 (AEG-1) is one of found in the brain tissue of HIV patients with dementia. In recent years, studies have shown that AEG-1 regulates a variety of central nervous system diseases, but its study on learning and cognition has not been reported. Hippocampus and cortex play an important role in learning and cognition. In this paper, CRISPR/Cas9 technology combined with Cre/loxp system was used to construct hippocampal cortex specific AEG-1 knockout mice, and the correlation between AEG-1 and learning cognition was preliminarily studied on the basis of this model mouse. Firstly, flox homozygous AEG-1^fl/fl mice inserted into the loxp site were constructed, and bred with hippocampal cortex specific Cre^+/+ recombinase expressing tool mice. Secondly, hippocampal cortex specific AEG-1 knockout mice with AEG-1 ^fl/fl Cre⁺ were selected by PCR. Thirdly,Western blot and immunofluorescence were used to detect the knockout efficiency of AEG-1 gene in the hippocampus and cortex. Last but not least, the new object recognition box and 3-chambered social interaction box combined with SMART 3.0 analysis system were used to preliminarily evaluate the learning memory and social interaction behavior of hippocampal cortico-specific AEG-1 knockout mice. Results: The knockout mice with AEG-1 ^fl/fl Cre⁺ were successfully obtained.AEG-1 protein expression in hippocampus and cortex of mice was significantly lower than that in control group. The new object recognition results showed that the discriminant coefficient of AEG-1 knockout mice was significantly lower than that of the control group, indicating that the learning and memory ability of AEG-1 knockout mice was weak. However, the social interaction showed that there was no significant difference in social interaction between AEG-1 knockout mice and the control group. These results lay the foundation for future studies on AEG-1 in learning cognition.