Generation of shRNAs from randomized oligonucleotides

Abstract

Suppression of gene expression by small interfering RNA (siRNA) has proved to be a gene-specific and cost effective alternative to other gene suppression technologies. Short hairpin RNAs (shRNAs) generated from the vector-based expression are believed to be processed into functional siRNAs in vivo, leading to gene silencing. Since an shRNA library carries a large pool of potential siRNAs, such a library makes it possible to knock down gene expression at the genome wide scale. Although much of research has been focused on generating shRNA libraries from either individually made gene specific sequences or cDNA libraries, there is no report on constructing randomized shRNA libraries, which could provide a good alternative to these existing libraries. We have developed a method of constructing shRNAs from randomized oligonucleotides. Through this method, one can generate a partially or fully randomized shRNA library for various functional analyses. We validated this procedure by constructing a p53-specific shRNA. Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells. We then made a partially randomized shRNA library. Sequencing of 15 randomly picked cloned confirmed the randomness of the library. Therefore, the library can be used for various functional assays, such as target validation when a suitable screening or selection method is available.

Fig. 1

Illustration of cloning strategy. A: Incorporation of 19 randomized nucleotides into PCR products. PCR reactions were performed using the cloned H1 promoter as a template and primers H1-5.1 and H1-R-3.2. B: Ligation of siRNA-loop-1. After PCR, the PCR product was digested with Bcc I and then ligated to siRNA-loop-1. C: Generation of palindromic sequences. Digestion with the nicking enzyme N.Alw I generated a partially single-stranded and double-stranded structure. At 72°C Taq polymerase converted the single-stranded DNA to double stranded DNA. D: Cloning. The extended product was digested with BamH I and then ligated to pSK-H1-Pu-X that had been digested with BamH I and Xcm I.

Fig. 2

Generation of p53-shRNA. The same strategy described in Fig. 1 was used to construct p53-shRNA. A: PCR and Bcc I digestion. PCR amplification yielded a 150 bp band (lane 1) and digestion of the PCR product with Bcc I shifted the band to 130 bp (lane 2). B: Ligation. Bcc I-digested DNA fragment was ligated to siRNA-loop-1 at 4°C overnight. Lane 3, before ligation; lane 4, after ligation. C: Extension. Ligated DNA fragment was treated with the nicking enzyme N.Alw I and then extended by Taq polymerase in the presence of dNTPs. Lanes 5, before nicking; 6, after nicking; 7, after extension. The extended product was about 160 bp. D: Cloning. Plasmid DNA was isolated from five positive clones and then digested with BamH I and Kpn I. M, 25 bp DNA ladders. All gels are 2%.

Fig. 3

Suppression of p53 by p53-shRNA. A: Detection of p53 expression by Western blot. Plasmid carrying an appropriate DNA fragment was introduced into MCF-7 cells. Proteins samples were prepared as described in Materials and Methods. Lanes 1, vector control; 2, p53-shRNA/pSK-H1-Pu-X; 3, p53-shRNA-p (positive control) and 4, p53-shRNA/pSK-H1-Pu-X/Xho I. ?-actin serves as a loading control. B: Removal of siRNA-loop-1 in p53-shRNA. Both constructs were digested with BamH I and Kpn I. M, 25 bp DNA ladders; lanes 1, p53-shRNA/pSK-H1-Pu-X; 2, p53-shRNA/pSK-H1-Pu-X/Xho I. C: Sequence comparison of p53-shRNA/pSK-H1-Pu-X and p53-shRNA/pSK-H1-Pu-X/Xho I. The p53 specific sequence is underlined.

Fig. 4

Sequences of 15 randomly picked clones from the library. N=A or G or C or T; H = A or C or T; W = A or T.