摘要
目的构建敲减小鼠海马神经元兴奋性氨基酸转运体3(excitatory amino acid transporter 3,EAAT3)重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体,建立一种小鼠海马EAAT3敲减动物模型。方法基于SLC1A1(solute carrierfamily 1 member 1)基因mRNA 序列设计4条shRNA(short hairpin RNA),将其模板DNA与酶切后的AAV-GP-1(pAAV-U6-EGFP)载体质粒连接、转化并测序鉴定得到重组质粒,将重组质粒、pHelper包装质粒和pAAV-DJ辅助质粒共同转染至AAV-293细胞包装得到rAAV;用所得rAAV侵染HT-22细胞,72h后RT-qPCR检测SLC1A1mRNA表达水平;36只成年C57小鼠随机分为6组,每组6只,予海马注射rAAV-shRNA-SLC1A1-2-EGFP病毒液1μL/侧,分别于注射后即刻、24h、7d、14d、21d、28d取双侧海马组织,Westernblot检测EAAT3表达水平。结果经测序重组质粒核苷酸序列正确,包装所得病毒浓缩滴度为1×1013TU/ml;rAAV感染HT-22细胞72h后SLC1A1表达量明显降低且显著低于阴性对照(P<0.01,n=3);小鼠海马注射rAAV-shRNA-SLC1A1-2-EGFP7d后EAAT3相对表达量下降(P<0.01),注射后21d、28d表达进一步降低(P<0.01)。结论成功构建介导shRNA表达的重组腺相关病毒系统,建立了一种有效的小鼠海马EAAT3敲减动物模型。
Objective To construct a recombinant adeno-associated virus vector to interfere with the expression of EAAT3 in hippocampus cells of mice and establish an animal model for EAAT3 knockdown in mouse hippocampus. Methods Based on the SLC1A1mRNA sequence, four shRNA sequences which interfered with the expression of gene SLC1A1 were designed and then ligated into the vector plasmid AAV-GP-1 (pAAV-U6-EGFP) after enzymes restriction to obtain the recombinant plasmids. The recombinant plasmids were transfected into AAV-293 cells with pHelper packaged plasmids and pAAV-DJ assistant plasmids to get asrAAV for titers identification. The rAAV suspension was transfected into HT-22 cells, and the expression of EAAT3 was detected by RT-qPCR 72 h later. Thirty-six adult C57 mice were randomly divided into 6 groups, with 6 mice in each group. rAAVshRNA- SLC1A1-2-EGFP virus solution (1 μl) was injected into each side of hippocampus. The bilateral hippocampus tissues were taken immediately and at 24 h, 7 d, 14 d, 21 d, 28 d after injection. Then the expression level of EAAT3 was detected by western blot. Results Nucleotide sequence of rAAV-shRNA-SLC1A1 was correct identified by sequencing, and the virus concentration titer obtained from the package was 1×1013 TU/ml. Compared with the control group and NC group, the mRNA expression of SLC1A1 gene significantly declined at 72 h in HT-22 cells (P =0.000). The expression level of EAAT3 declined significantly at 7 d (P <0.001), and further decline was still observed at 21 d and 28 d after injection (P =0.000, respectively). Conclusion A system of recombinant adeno-associated virus inducing a shRNA expression and a hippocampal EAAT3 knockdown animal model in mice have been established successfully.
作者
侯爱生
王晓燕
武屹爽
曹福羊
刘蔷薇
张璇
沈浩
曹江北
HOU Aisheng;WANG Xiaoyan;WU Yishuang;CAO Fuyang;LIU Qiangwei;ZHANG Xuan;SHEN Hao;CAO Jiangbei(Anesthesia and Operation Center,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China;Department of Anesthesia,the Fourth Medical Center,Chinese PLA General Hospital,Beijing 100037,China)
出处
《解放军医学院学报》
CAS
2019年第5期473-477,481,共6页
Academic Journal of Chinese PLA Medical School
基金
国家自然科学基金项目(81771129)~~
关键词
腺相关病毒
兴奋性氨基酸转运体3
短发卡RNA
小鼠
海马
adeno-associated virus
excitatory amino acid transporter
short hairpin RNA
mouse
hippocampus
