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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. 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.
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f3: 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.

Mentions: To determine whether the p53-shRNA construct (p53-shRNA/pSK-H1-Pu-X) can generate functional siRNA to suppress p53 expression, we introduced p53-shRNA/pSK-H1-Pu-X into MCF-7 cells that express wild type p53 (23). We also made a p53 shRNA construct using the same p53 specific sequence based on pSUPER vector previously described (8), to serve as a positive control (p53-shRNA-p).We found that p53-shRNA/pSK-H1-Pu-X was able to inhibit the endogenous p53 expression, with over 80% suppression compared to the vector control (Fig. 3, lane 2). This was about comparable to that of the positive control p53-shRNA-p (Fig. 2, lane 3), suggesting that the p53 shRNA constructed through this presently described approach is functional.


Generation of shRNAs from randomized oligonucleotides.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211575&req=5

f3: 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.
Mentions: To determine whether the p53-shRNA construct (p53-shRNA/pSK-H1-Pu-X) can generate functional siRNA to suppress p53 expression, we introduced p53-shRNA/pSK-H1-Pu-X into MCF-7 cells that express wild type p53 (23). We also made a p53 shRNA construct using the same p53 specific sequence based on pSUPER vector previously described (8), to serve as a positive control (p53-shRNA-p).We found that p53-shRNA/pSK-H1-Pu-X was able to inhibit the endogenous p53 expression, with over 80% suppression compared to the vector control (Fig. 3, lane 2). This was about comparable to that of the positive control p53-shRNA-p (Fig. 2, lane 3), suggesting that the p53 shRNA constructed through this presently described approach is functional.

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