Department of Genetics, Hebei Key Lab of Laboratory Animal, Hebei Medical University, Shijiazhuang, 050017, Hebei Province, China.
School of Stomatology, Hebei Medical University, Shijiazhuang, 050017, Hebei Province, China.
Microb Cell Fact. 2017 Oct 30;16(1):183. doi: 10.1186/s12934-017-0800-z.
Exogenous RNAs can specifically up-regulate or down-regulate gene expression after they enter into cells. Alu RNAs are the main constituent of human transcriptome and participate in gene expression regulation. AluY elements belong to a subfamily of Alus and are the youngest Alus. In this paper, we established the technology method of preparing genetically engineered humanized AluY RNAs (AluY RNAs) from Escherichia coli (E. coli) strains. This technology method also can be used to prepare other genetically engineered humanized RNAs that can be used for cytology experiments.
Different copies of human AluY elements were inserted into pET-28α plasmid (pET) to construct pET-AluY plasmids that were transformed into BMBL21-DE3 (DE3) E. coli. Isopropylthio-β-D-galactoside (IPTG) induction inhibited transformed bacterial growth after DE3 E. coli were transformed by pET-AluY × 8 plasmid (8 copies of AluYs were inserted into pET); northern blotting was used to detect the amount of AluY RNAs after 2, 4, 6, 8, 10, 12, 14 and 16 h inducing with IPTG. The results showed that the amount of AluY RNAs was the highest at 4 h; 1, 2, 4, 8 or 14 copies of AluY elements were inserted into the pET to construct pET-AluY plasmids that were transformed into DE3 bacteria, the northern blotting results showed that AluY RNAs production amount increased with the increase of AluY copy number; pET-AluY × 8 DE3 bacteria did not produce AluY RNAs without IPTG induction, AluY RNA production kept similar when inducing by 0.1-0.4 mg/ml IPTG induction, however, AluY RNA production slightly decreased if deviating from the above concentration range; pET-AluY × 8 DE3 bacteria were cultured at 34, 37 or 40 °C and the results showed that AluY RNA production was the highest under 37 °C cultivation; pET-AluY × 8 plasmid was transformed into three kinds of BL21 bacteria, including DE3, BMBL21-DE3-pLysS (pLysS) and Trans BL 21 (TransBL), the results showed that AluY RNA production was the highest when using DE3 bacteria.
The optimal conditions of producing AluY RNAs were: a kind of host bacteria of DE3, an engineering bacteria concentration of OD 1.0, an IPTG concentration of 0.2 mg/ml, a culturing temperature of 37 °C and a culturing time of 4 h. Pure AluY RNAs occupied 15.8% of extractive total RNAs and the mean yield of pure AluY RNAs in 100 ml bacteria solution was 0.46 mg.
外源性 RNA 进入细胞后可特异性地上调或下调基因表达。Alu RNA 是人类转录组的主要组成部分，并参与基因表达调控。AluY 元件属于 Alu 家族的一个亚家族，是最年轻的 Alu。本文建立了从大肠杆菌（E. coli）菌株中制备基因工程人源化 AluY RNA（AluY RNA）的技术方法。该技术方法还可用于制备其他可用于细胞学实验的基因工程人源化 RNA。
将不同拷贝的人类 AluY 元件插入 pET-28α 质粒（pET）中，构建 pET-AluY 质粒，转化至 BMBL21-DE3（DE3）大肠杆菌中。异丙基-β-D-硫代半乳糖苷（IPTG）诱导抑制了转化细菌的生长，之后将 pET-AluY×8 质粒（8 个 AluYs 插入 pET）转化至 DE3 大肠杆菌；通过Northern 印迹检测诱导 2、4、6、8、10、12、14 和 16 h 后 AluY RNA 的量。结果表明，诱导 4 h 后 AluY RNA 的量最高；将 1、2、4、8 或 14 个 AluY 元件插入 pET 构建 pET-AluY 质粒，转化至 DE3 细菌中，Northern 印迹结果表明，AluY RNA 的产量随着 AluY 拷贝数的增加而增加；在没有 IPTG 诱导的情况下，pET-AluY×8 DE3 细菌不产生 AluY RNA，当用 0.1-0.4 mg/ml IPTG 诱导时，AluY RNA 的产量保持相似，然而，如果偏离上述浓度范围，AluY RNA 的产量会略有下降；在 34、37 或 40°C 下培养 pET-AluY×8 DE3 细菌，结果表明 37°C 培养时 AluY RNA 的产量最高；将 pET-AluY×8 质粒转化至三种 BL21 细菌，包括 DE3、BMBL21-DE3-pLysS（pLysS）和 Trans BL 21（TransBL），结果表明使用 DE3 细菌时 AluY RNA 的产量最高。
产生 AluY RNA 的最佳条件为：宿主菌为 DE3，工程菌浓度 OD1.0，IPTG 浓度 0.2 mg/ml，培养温度 37°C，培养时间 4 h。提取总 RNA 中纯 AluY RNA 占 15.8%，100 ml 细菌液中纯 AluY RNA 的平均产量为 0.46 mg。
