牛lnc RNA SNHG12过表达载体构建

Construction of Lnc RNA SNHG12 overexpression vector

中文关键词: pcDNA3.1-EGFP-SNHG12  荧光定量技术  CART  细胞转染

英文关键词: pcDNA3.1-EGFP-SNHG12  Fluorescence quantitative technique  The CART  Cell transfection

中文摘要:

      目的 构建牛lncRNA SNHG12过表达重组载体，为进一步探究lncRNA SNHG12、miRNA 491和牛CART基因三者的互作关系奠定试验基础。方法 美国国家生物技术信息中心（national center for biotechnology information, NCBI）基因数据库（登录号：NC_037329.1）获取牛lncRNA SNHG12序列，经聚合酶链式反应（PCR）扩增，双酶切后连接pcDNA3.1-EGFP载体得到重组质粒pcDNA3.1-EGFP-SNHG12。将pcDNA3.1-EGFP-SNHG12、miRNA 491和牛CART三种质粒为一组转染293T细胞，利用荧光定量PCR(quantitative real-time PCR, qRT-PCR)检测lncRNA SNHG12的表达水平。结果 显示pcDNA3.1-EGFP-SNHG12载体序列正确；转染293T细胞绿色荧光量达50%左右，且强度适中，说明转染效果良好。荧光定量PCR结果显示在293T细胞中NC组的lncRNA SNHG12的表达水平极显著高于miRNA 491组。结论 pcDNA3.1-EGFP-SNHG12载体构建成功，并且lncRNA SNHG12与miRNA491具有互作作用。本试验将为后续探究lncRNA SNHG12、miRNA 491、牛CART基因三者网络关系创造试验条件。

英文摘要:

      Objective To construct lncRNA SNHG12 overexpression recombinant vector, and to lay the experimental foundation for further exploring the interaction between lncRNA SNHG12, miRNA 491 and CART gene. Methods national center for biotechnology information (NCBI) Gene Database (No. : NC_037329.1) lncRNA SNHG12 sequence was obtained and amplified by polymerase chain reaction (PCR). After double digestion, pcDNA3.1-EGFP vector was ligated to obtain recombinant plasmid PCDNA3.1-EGFP-SnHG12. The pcDNA3.1-EGFP-SNHG12, miRNA 491 and CART plasmids were transfected into 293T cells, and the expression level of lncRNA SNHG12 was detected by quantitative real-time PCR(qRT-PCR). The results showed that the sequence of pcDNA3.1-EGFP-SNHG12 vector was correct. The green fluorescence amount of 293T cells transfected was about 50%, and the intensity was moderate, indicating that the transfection effect was good. qRT-PCR results showed that the expression level of lncRNA SNHG12 in NC group was significantly higher than that in miRNA 491 group in 293T cells. Conclusion pcDNA3.1-EGFP-SNHG12 vector was successfully constructed, and lncRNA SNHG12 had interaction with miRNA491. This study will create experimental conditions for further exploring the network relationship among lncRNA SNHG12, miRNA 491 and CART genes.