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

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。

Abstract:

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.