引用本文:张中卒1,黄 路2,虞志明1,张 政3,韩智敏1,杨 东1,詹 平1,曹 凯1.Let-7a稳定转染尤文肉瘤细胞系的建立[J].重庆医科大学学报,2013,(11):1249~1106
Let-7a稳定转染尤文肉瘤细胞系的建立
Establishment of Ewing’s sarcoma cell lines stably expressing let-7a
DOI:
中文关键词:  let-7a  尤文肉瘤  A673细胞  SK-ES-1细胞  慢病毒
英文关键词:let-7a  Ewing’s sarcoma  A673 cells  SK-ES-1 cells  lentivirus
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作者单位
张中卒1,黄 路2,虞志明1,张 政3,韩智敏1,杨 东1,詹 平1,曹 凯1 1. 南昌大学第一附属医院骨科南昌 3300062. 江西省妇幼保健院儿童保健科南昌 3300063. 重庆医科大学基础医学院生物与遗传学教研室重庆 400016 
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中文摘要:
      目的:构建携带let-7a基因的真核表达载体,建立稳定转染该质粒的尤文肉瘤(Ewing’s sarcoma,ES)细胞株A673/let-7a、SK-ES-1/let-7a。方法:通过 Gateway克隆技术,扩增attB1-K-eGFP-let-7a-1-attB2片段,与pDONR221进行BP重组反应产生入门克隆,再与母载体———pLV.Des2d.P/neo进行LR重组反应,生成目的质粒———pLV.Des2d.P/neo-EF1A>eGFP/let-7a;通过PCR及测序验证目的质粒;将携带let-7a的重组慢病毒质粒稳定转染ES细胞株 A673和 SK-ES-1获得A673/let-7a、SK-ES-1/let-7a细胞株;通过real-time PCR检测稳定转染细胞株内 let-7a的表达水平,Western blot检测其靶基因CDK6的表达。结果:PCR证实获得的目的条带与理论值相符,测序分析证实携带 let-7a 的真核表达载体插入序列及位点正确;real-time PCR及Western blot检测结果显示,与转染空载质粒及未处理组的 A673、SK-ES-1细胞株相比,稳定转染重组目的基因的A673/let-7a、SK-ES-1/let-7a细胞高表达let-7a,分别达到8.5倍(P=0.000)和6.5倍(P=0.001),差异具有统计学意义;且细胞内let-7a靶基因CDK6的表达量明显减少。结论:成功构建稳定表达let-7a的ES细胞株 A673/let-7a、SK-ES-1/let-7a。
英文摘要:
      Objective:To construct a eukaryotic expression plasmid carrying let-7a and to establish the Ewing’s sarcoma(ES) cell line A673 and SK-ES-1 stably expressing let-7a. Methods:Fragment of attB1-K- eGFP-let-7a-1-attB2 coding let-7a was amplified using Gateway protocols. The let-7a gene was recombined with donor vector pDONR221 to construct an entry plasmid pDONR221-eGFP-let-7a,which forwarded to recombine with pLV.Des2d.P/neo to reconstruct pLV.Des2d.P/neo-EF1A>eGFP/Let-7a. PCR and se-quencing were used to confirm the plasmid. Lenti-let-7a transfected ES cell lines A673/let-7a and SK-ES-1/let-7a which can sta-bly express let-7a were screened out. A673/let-7a and SK-ES-1/let-7a were confirmed by real-time PCR. Western blot analysis was performed to detect the expression of CDK6 in A673/let-7a and SK-ES-1/let-7a cells,which was the target gene of let-7a. Results:PCR results confirmed that the value of target fragment was consistent with the theoretic value. Sequencing analysis showed that let-7a was correctly inserted into the pLV.Des2d.P/neo-EF1A>eGFP/let-7a plasmid. Real-time PCR indicated that the expression of let-7a was increased remarkably in A673/let-7a and SK-ES-1/let-7a compared with that of control and blank cells;expressing values of let-7a were as high as 8.5 folds(P=0.000) and 6.5 folds(P=0.001) in A673/let-7a and SK-ES-1/let-7a respectively. Western blot analysis showed that ectopic expression of let-7a down-regulated the expression of CDK6. Conclusions:ES cell lines A673/let-7a and SK-ES-1/let-7a which can stably express let-7a are successfully established.
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