YIN Shu-xian, ZHAO Yue-hua, LIU Chao, LV Zhan-jun, WANG Xiu-fang
Objective: Exogenous RNAs can specifically up-regulate or down-regulate gene expression after they enter into cells. The preparation methods of exogenous RNAs mainly include chemical synthesis, in vitro transcription and extraction from cells. Alu DNA and Alu RNA are the main constituents of human genome and transcriptome and participate in gene expression regulation. The technology method of preparing genetically engineered humanized Alu RNA (Alu RNA) from engineering bacteria was established. The extracted RNAs using this technology method can satisfy the requirement of general biology experiments. Methods and results:Different copies of human Alu elements were inserted into pET-28α plasmid (pET) to construct pET-Alu plasmids that were transformed into BL-21 bacteria. The effects of different conditions on the Alu RNA production were explored. Isopropylthio-β-d-galactoside (IPTG) induction inhibited transformed bacterial growth after BMBL-21 (DE3) competent cells (DE3 cells) were transformed by pET-Alu×8 plasmid (8 copies of Alus were inserted into pET); Northern blotting was used to detect the amount of Alu RNA after 2, 4, 6, 8, 10, 12, 14 and 16 hours inducing with IPTG. The results showed that the amount of Alu RNA was highest at 4 hours; 1, 2, 4, 8 or 14 copies of Alu elements were inserted into the pET to construct pET-Alu plasmids that were transformed into DE3 bacteria, the Northern blotting results showed that Alu RNA production amount increased with the increase of Alu copy number; pET-Alu×8 DE3 bacteria did not produce Alu RNA without IPTG induction, Alu RNA production kept similar when inducing by 0.1mg/ml to 0.4mg/ml IPTG induction, however, Alu RNA production slightly decreased if deviating from the above concentration range; pET-Alu×8 DE3 bacteria were cultured at 34℃, 37℃ or 40℃ and then were induced by IPTG for 4 hours, the results showed that, under the condition of 37℃ cultivation, Alu RNA production was the highest; pET-Alu×8 plasmid was transformed into three kinds of BL-21 cells, including DE3, BMBL21-DE3-pLysS (pLysS) and Trans BL 21 (TransBL), the results showed that Alu RNA production was the highest when using pET-Alu×8 DE3. Conclusion: The preparation technology of genetically engineered humanized Alu RNA was established. The highest production of Alu RNA can be obtained based on the following conditions:pET-Alu×14 plasmid was transformed into DE3 bacteria; the transformed bacteria were cultured at 37℃ to OD 1.0 at 600nm, and then were induced by 0.2mg/ml IPTG for 4 hours. Pure Alu RNA occupies 15.8% of extractive RNA and the mean yield of pure Alu RNA in 100ml bacteria solution is 0.46mg.
