shRNA表达载体构建方法的优化

The Optimization of Short Hairpin RNA Expression Vector Construction

目的探讨shRNA表达载体的构建方法 ,以加速RNA干扰研究的进程。方法对shRNA表达载体的构建过程进行分析和监测 ,并加以优化。结果发现shRNA表达载体构建的退火过程容易产生障碍 ,经优化退火缓冲液的NaCl含量后 ,能明显提高退火效率及shRNA表达载体构建的成功率。结论shRNA表达载体构建的退火过程需加以关注 ,退火缓冲液中NaCl含量应提高至 2 0

Objective To improve the efficiency of short hairpin RNA (shRNA) expression vector construction, so that RNA interference technology can be more broadly used in biomedical research, as well as in the development of novel therapeutics. Methods The annealing conditions of the oligonucleotide cassettes were analyzed and optimized in order to achieve the most efficient construction of shRNA expressing vectors. Results We found that the annealing step of the oligonucleotide cassettes was critical to the efficient construction of shRNA expression vectors. Better annealing and cloning efficiency was achieved by adjusting the concentration of NaCl in the annealing buffer to 200mmol/L. Conclusion Our results suggest that attentions should be paid to the oligonucleotide cassette annealing conditions during of the construction of shRNA expression vectors.