|本期目录/Table of Contents|

[1]尹磊,王成泉,王珺,等.Per2-shRNA慢病毒构建及嘌呤霉素筛选Per2低表达的细胞株[J].宁夏医科大学学报,2014,(03):252-255.
 YIN Lei,WANG Chengquan,WANG Jun,et al.Constructing Per2-shRNA Lentiviral and Selecting Low-expression Cell Lines of Per2 with Puromycin[J].Ningxia Medical University,2014,(03):252-255.
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Per2-shRNA慢病毒构建及嘌呤霉素筛选Per2低表达的细胞株(PDF)
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《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2014年03期
页码:
252-255
栏目:
论著
出版日期:
2014-03-30

文章信息/Info

Title:
Constructing Per2-shRNA Lentiviral and Selecting Low-expression Cell Lines of Per2 with Puromycin
作者:
尹磊1王成泉1王珺1夏鹤春2
1.宁夏医科大学,银川 750004; 2.宁夏医科大学总医院神经外科,银川 750004
Author(s):
YIN Lei1WANG Chengquan1WANG Jun1XIA Hechun2
1.Ningxia Medical University; 2.Dept. of Neurosurgery, the General Hospital of Ningxia Medical University, Yinchuan 750004
关键词:
Period2基因shRNAU343细胞慢病毒
Keywords:
Per2 gene shRNA U343 cells lentiviral
分类号:
Q78
DOI:
-
文献标志码:
A
摘要:
目的构建人Period2(Per2)基因shRNA慢病毒。方法设计并合成3对特异性人Per2基因shRNA靶序列,构建到pENTRTM/U6(GV112)慢病毒载体中,包装慢病毒,用real-timePCR筛选取最佳片段。对感染的U343细胞,经嘌呤霉素初步筛选,Westernblot鉴定Per2蛋白表达,得到Per2低表达的细胞。结果成功构建了具有Per2沉默效应的shRNA慢病毒。结论此次包装的慢病毒可以成功抑制Per2基因在胶质瘤U343细胞中的表达,并可用嘌呤霉素筛选低表达细胞株。
Abstract:
Objective To construct shRNA lentiviral vectors which can interfere expression of Period2(Per2)gene, and to establish glioma U343 cell lines with a low expression of Per2 gene. Methods Three pairs of specific human Per2 gene shRNA target sequence were designed and synthesized, which could link with pENTRTM/U6(GV112)lentiviral vector after annealing. Screening single colonies, then plasmid were extracted for PCR and sequencing. The plasmids were transfected into 293T cells, and the viral supernatant was collected to detect the titer of viral. Real-time PCR was used for selecting the best jamming effect of the lentivirus infection U343 cells, then getting cell lines with a low expression of Per2 by puromycin, Western blot was used for detecting the effects of interference. Results The results of PCR and DNA sequencing showed that knock-down of Per2 was successfully constructed with interference effects by shRNA lentiviral vectors; filter out the best sequence for Interference by real-time PCR, then the drug screening method was used for the determination of viral and the titer was 6.0×108 Tu·mL-1; Western blot validated shRNA lentiviral infection after glioma U343 cells Per2 protein expression levels were significantly decreased. Conclusion Lentiviral-mediated shRNA interference can be effectively suppressed Per2 gene in glioma U343 cells for further in-depth study and research Per2 association between glioma experimental basis.

参考文献/References:

[1] Hua H,Wang Y,Wan C,et al.Circadian gene mPer2 overexpression induces cancer cell apoptosis[J].Cancer Sci,2006,97(7):589-596.
[2] Sherry L Winter,Lucine Bosnoyan-Collins,Dushanthi Pinnaduwage,et al. Expression of the circadian clock genes Per1 and Per2 in sporadic and familial breast tumors[J].Neoplasia,2007,9(10):797-800.
[3] Zeman M,Vician M,Monosíková J,et al. Deregulated expression of the per2 gene in human colorectal carcinoma[J].Mol Med Rep,2008,1(4):599-603.
[4] 池闯,何志锋,刘瑜,等.生物钟基因Per2在非小细胞肺癌中的表达及其临床意义[J].中华肿瘤杂志,2013,,35(2):129-31.
[5] Takahiro Nagase,Ken-ichi Ishikawa.Prediction of the Coding Sequences of Unidentified Human Genes. VII. The Complete Sequences of 100 New cDNA Clones from Brain Which Can Code for Large Proteins in vitro[J].DNA Res,1997,4:141-150.
[6] XH chun,NZ feng,HS Cai,et al. Deregulated Expression of the Per1 and Per2 in Human Gliomas[J].Can J Neurol Sci,2010,37:365-370.
[7] 王凡,陈治军,夏鹤春,等.胶质瘤组织中Per2和PCNA的表达及其相关性[J].山东医药杂志,2012,26(52):8-9
[8] Virchow C,Szczeklik A,Bianco S. Intolerance to tartrazine in aspirin-induced asthma:results of multicenter study[J].Respiration,1988,53:20-23.
[9] 邱志兵,陈鑫,段超,等.靶向 STAT3 的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响[J].中华实验外科杂志,2011,28(4):506-509.

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备注/Memo

备注/Memo:
收稿日期:2013-10-06 基金项目:国家自然科学基金(81160313) 作者简介:尹磊(1985-),女,河北人,在读硕士研究生,从事胶质瘤研究。 通信作者:夏鹤春。E-mail:xhechun@aliyun.com
更新日期/Last Update: 2014-03-20