Abstract
Small interference RNAs (siRNA) have been shown to be useful in the field of gene therapy and gene function studies. As a siRNA expression vector, pSilencer employ RNA polymerase III promoters and could stably produce siRNA for weeks. But once one siRNA sequence was inserted into the pSilencer vector, the other siRNA sequence will hardly be reconstructed, because the site of siRNA production has been occupied and difficult to be changed, so it is not suitable for screen of effective siRNA sequence. To solve this problem, we constructed the subclone pSilcencer329, which generated from pSilencer3.1, then developed a PCR based method of constructing siRNA expression vectors, and generated pSilencerBCL2L2 recombinants efficiently. This method was proven to be effective, reliable, and less expensive, and thus will be of great help in regular gene silencing studies, and will be especially suitable for large scale gene function analysis.
Similar content being viewed by others
References
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Scherr M, Eder M (2007) Gene silencing by small regulatory RNAs in mammalian cells. Cell Cycle 6:444–449
Du Y, Yin F, Liu C et al (2006) Depression of MAD2 inhibits apoptosis of gastric cancer cells by upregulating Bcl-2 and interfering mitochondrion pathway. Biochem Biophys Res Commun 345:1092–1098
Liang X, Liu Y, Zhang Q et al (2007) Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax. J Immunol 178:503–510
Bantounas I, Phylactou LA, Uney JB (2004) RNA interference and the use of small interfering RNA to study gene function in mammalian systems. J Mol Endocrinol 33:545–557
Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59:75–86
Luo Q, Kang Q, Song WX et al (2007) Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. Gene 395:160–169
Du C, Niu R, Chu E, Zhang P, Lin X (2006) Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio. J Biochem (Tokyo) 139:913–920
Pei Y, Tuschl T (2006) On the art of identifying effective and specific siRNAs. Nat Methods 3:670–676
Carvalho G, Fabre C, Braun T et al (2007) Inhibition of NEMO, the regulatory subunit of the IKK complex, induces apoptosis in high-risk myelodysplastic syndrome and acute myeloid leukemia. Oncogene 26:2299–2307
Ui-Tei K, Naito Y, Saigo K (2007) Guidelines for the selection of effective short-interfering RNA sequences for functional genomics. Methods Mol Biol 361:201–216
Birmingham A, Anderson E, Sullivan K et al (2007) A protocol for designing siRNAs with high functionality and specificity. Nat Protoc 2:2068–2078
Acknowledgments
This work was supported by grant from the Natural Science Foundation of China (No.30700357).
Author information
Authors and Affiliations
Corresponding author
Additional information
Zhiyong Zhang and Lihui Han contributed equally to this work.
Rights and permissions
About this article
Cite this article
Zhang, Z., Han, L., Liang, X. et al. A PCR based method to construct small interference RNA expression vectors. Mol Biol Rep 36, 801–805 (2009). https://doi.org/10.1007/s11033-008-9248-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-008-9248-x