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

doi:10.13523/j.cb.2008128

China Biotechnology

2020, Vol. 40

Issue (11): 10-20 DOI: 10.13523/j.cb.2008128

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

YU Chun-yang¹,ZHANG Chun¹,GUO Le³,WAN Pan-pan¹,HUANG Yue⁴,WANG Feng^1,^5**(

),LIU Kun-mei^1,^2**(

)

1 Ningxia Key Laboratory of Cerebrocranial Diseases, Yinchuan 750004, China
2 Medical Science Research Institution of Ningxia Hui Autonomous Region, Yinchuan 750004, China
3 Clinical Medical College of Ningxia Medical University, Yinchuan 750004, China
4 School of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
5 Department of Neurosurgery, General Hospital of Ningxia Medical University, Yinchuan 750004, China

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Abstract

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.

Key words： AEG-1 Cre/loxp system Conditional knockout Learning and cognition

Received: 20 August 2020 Published: 11 December 2020

ZTFLH:

Q819

Corresponding Authors: Feng WANG,Kun-mei LIU E-mail: nxwwang@163.com;lkm198507@126.com

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	Chun-yang YU
	Chun ZHANG
	Le GUO
	Pan-pan WAN
	Yue HUANG
	Feng WANG
	Kun-mei LIU

Cite this article:

YU Chun-yang,ZHANG Chun,GUO Le,WAN Pan-pan,HUANG Yue,WANG Feng,LIU Kun-mei. Construction of Hippocampal Cortical Specific Knockout AEG-1 Gene Mice and Preliminary Study on Its Behavior. China Biotechnology, 2020, 40(11): 10-20.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2008128 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I11/10

Fig.1 Constructive strategy of AEG-1 flox knockout mice (a) Donor vector and CRISPR/Cas9 system (b)Targeted vector

Fig.2 Schematic design of primers for AEG-1 flox mouse identification

Table 1 Primer information of AEG-1 flox mouse gene identification

Fig.3 Breeding flow chart of AEG-1^fl/flCre⁺

Table 2 Information of primers for AEG-1^fl/wt Cre⁺ mouse

Fig.4 Pattern diagram of novel object recognition

Fig.5 Pattern diagram of 3-chambered social test

Fig.6 PCR identification results of mice with AEG-1^{fl/ wt} genotype (a) PCR genotyping results of the primer 1 (b) PCR genotyping results of the primer 2 (c) PCR genotyping results of the primer 3 (d) PCR genotyping results of the primer 4 (e) PCR genotyping results of the primer 5 B6: Negative control of which template is genomic DNA of C57BL/6J mice; N: Blank control without template. TRANS 2K PLUS II marker size: 8 000bp, 5 000bp, 3 000bp, 2 000bp, 1 000bp, 750bp, 500bp, 250bp, 100bp

Fig.7 PCR identification results of mice with AEG-1^fl/flCre⁺ genotype (a) PCR genotyping results of the primer 1 (b) PCR genotyping results of the primer 2 (c) PCR genotyping results of the primer 3 Marker size: 700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp

Fig.8 Detection of expression of AEG-1 in the hippocampus and cortex by Western blot (a) AEG-1 and GAPDH protein were detected by western blot (b) Quantitative data of densitometric analyses The ratio of AEG-1 protein levels to GAPDH were displayed as mean ± SD, n=3, *** P<0.001 vs. wt group. H: Hippocampus; C: Cortex; KO: Knock out

Fig.9 Detection of expression of AEG-1 in the hippocampus and cortex by immunofluorescence (a-d) The quantity of AEG-1 specific neurocytes expression in AEG-1 KO homozygous mice was significantly reduced compared with wt mice in the hippocampal CA1 region (a), CA3 region (b), DG region (c) and cortical region (d) (e-h) Bar charts compare the numbers of AEG-1 KO neurocytes to the wt in the CA1(e), CA3(f), DG(g) districts of the hippocampus and in cortex(h) LAS X software analyzed the results of KO group **** P<0.000 1 vs wt group (Scale bar:25μm)

Fig.10 Detection of learning cognitive ability of AEG-1 KO mice by novel object recognition

Fig.11 Detection of social interaction ability of AEG-1 KO mice by 3-chambered social test


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