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Generation of shRNAs from randomized oligonucleotides.

Wu H, Dinh A, Mo YY - Biol Proced Online (2007)

Bottom Line: Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794 USA.

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.

No MeSH data available.


Related in: MedlinePlus

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%.
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f2: 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%.

Mentions: To determine whether functional shRNAs can be generated through this approach, we constructed a vector-based p53-shRNA following the strategy outlined in Fig. 1. To incorporate half of p53 shRNA palindromic sequence 5’-GACTCCAGTGGTAATCTAC (8) into PCR products, we used a sense primer, H1-5.1, and an antisense primer, p53-siRNA-3.1 (see Materials and Methods). Primer p53-siRNA-3.1 differed from R-H1-siRNA-3.2 in that the 19 base p53 sequence (8) replaced the 19 base randomized oligonucleotides of R-H1-siRNA-3.2. The amplified fragment was about 150 bp (Fig. 2A, lane 1). Following digestion with Bcc I, the digested fragment was 20 bp smaller than before digestion, as expected, resulting in a 130 bp fragment (Fig. 2A lane 2). After gel purification, the digested fragment was ligated to the 5’-phosphorylated siRNA-loop-1. Although ligation reactions are normally carried out at room temperature, or at 14-16°C, we carried the reaction out at 4°C overnight, due to the relatively small size of the loop. Under this condition, successful ligation was achieved because the 130 bp band was completely shifted to ~145 bp (Fig. 2B, lane 4). Since the Bcc I-digested fragment generated a G overhang at the digested end, this prevented end-end ligation of two molecules of the same species, thus, enhancing the ligation of siRNA-loop-1 and H1 promoter.


Generation of shRNAs from randomized oligonucleotides.

Wu H, Dinh A, Mo YY - Biol Proced Online (2007)

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%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211575&req=5

f2: 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%.
Mentions: To determine whether functional shRNAs can be generated through this approach, we constructed a vector-based p53-shRNA following the strategy outlined in Fig. 1. To incorporate half of p53 shRNA palindromic sequence 5’-GACTCCAGTGGTAATCTAC (8) into PCR products, we used a sense primer, H1-5.1, and an antisense primer, p53-siRNA-3.1 (see Materials and Methods). Primer p53-siRNA-3.1 differed from R-H1-siRNA-3.2 in that the 19 base p53 sequence (8) replaced the 19 base randomized oligonucleotides of R-H1-siRNA-3.2. The amplified fragment was about 150 bp (Fig. 2A, lane 1). Following digestion with Bcc I, the digested fragment was 20 bp smaller than before digestion, as expected, resulting in a 130 bp fragment (Fig. 2A lane 2). After gel purification, the digested fragment was ligated to the 5’-phosphorylated siRNA-loop-1. Although ligation reactions are normally carried out at room temperature, or at 14-16°C, we carried the reaction out at 4°C overnight, due to the relatively small size of the loop. Under this condition, successful ligation was achieved because the 130 bp band was completely shifted to ~145 bp (Fig. 2B, lane 4). Since the Bcc I-digested fragment generated a G overhang at the digested end, this prevented end-end ligation of two molecules of the same species, thus, enhancing the ligation of siRNA-loop-1 and H1 promoter.

Bottom Line: Western blot revealed that the p53-shRNA successfully suppressed expression of the endogenous p53 in MCF-7 cells.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794 USA.

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.

No MeSH data available.


Related in: MedlinePlus