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

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

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/flCre^+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/flCre^+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。

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/flmice 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/flCre^+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/flCre^+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.