Preparation of genetically engineered murine SINE RNA without endotoxin contamination.
Preparation of genetically engineered murine SINE RNA without endotoxin contamination.
复制标题
无内毒素污染的基因工程小鼠SINE RNA的制备
作者：
Liu X;Lv B;Yan L;Khan M;Ji N;Shah S;Song Z;Zhao Y;Su L;Wang X;Lv Z
SDS-NaCl过滤法的内毒素去除率高于SDS-NaCl离心法。通过掺入Triton X-114的SDS-NaCl过滤制备的RNA符合动物体内实验的要求。RNA在基因表达调控中发挥着重要作用，有望成为治疗癌症和老年性疾病的有效药物。在通过表达载体转化大肠杆菌后，通过SDS-NaCl离心提取RNA，这是获得基因工程RNA的方法。但该方法制备的RNA含有内毒素，限制了其在体内和细胞实验中的应用。本文在SDS-NaCl离心法的基础上，对SDS-NaCl过滤法进行了改进。SDS-NaCl过滤法的内毒素去除效率是SDS-NaCl离心法的近4.2倍。使用Triton X-114相分离进一步降低SDS-NaCl过滤提取的RNA的内毒素含量（从11.25 EU/µg RNA/ml降至0.08 EU/µg RNA/ml）。用本文建立的方法制备的RNA符合体内和细胞培养实验的要求。本文介绍了从pET-B1 as-DE 3 E中制备无内毒素B1 asRNA的方法。将含有串联SINE B1元件的pET-B1作为表达载体转化到大肠杆菌DE 3中，采用SDS-NaCl过滤结合Triton X-114相分离法进行纯化。
The endotoxin removal efficiency of SDS-NaCl filtration is higher than that of SDS-NaCl centrifugation. RNA prepared by SDS-NaCl filtration incorporating Triton X-114 meets the requirements for in vivo experiments on animals. RNAs have been elucidated to play the critical role in regulating gene expression and to be expected as effective drugs in the treatment of cancer and age-related diseases. RNAs are extracted by SDS-NaCl centrifugation after transformation of E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their application in vivo and in cell experments. Here we improved SDS-NaCl filtration method based on SDS-NaCl centrifugation method. Endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. Triton X-114 phase separation was used to reduce futher the endotoxin content of SDS-NaCI filtration-extracted RNA (from 11.25 EU/µg RNA/ml to 0.08 EU/µg RNA/ml). RNA prepared using the methods established in this paper meets the requirements for in vivo and cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 E. coli (DE3 transformed by pET-B1as expression vector which containing a tandem SINE B1 elements) using SDS-NaCl filtration incorporating Triton X-114 phase separation.
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DOI：
10.1016/j.btre.2016.10.001
发表时间：
2016-12
期刊：
Biotechnology reports (Amsterdam, Netherlands)
影响因子：
--
作者：
El-Ashram S;Al Nasr I;Suo X
通讯作者：
Suo X
影响因子：
3.8
作者：
Rozkov, Aleksei;Larsson, Bert;Schmidt, Stefan R.
通讯作者：
Schmidt, Stefan R.
DOI：
10.1126/science.aad8709
发表时间：
2016-06-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Sullenger BA;Nair S
通讯作者：
Nair S
影响因子：
5.6
作者：
Zhang Z;Ran Y;Shaw TS;Peng Y
通讯作者：
Peng Y
影响因子：
4.4
作者：
Ahn R;Gupta R;Lai K;Chopra N;Arron ST;Liao W
通讯作者：
Liao W