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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. 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.
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f1: 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.

Mentions: As shown in Fig. 1, the entire cloning procedure involves the following 4 steps.


Generation of shRNAs from randomized oligonucleotides.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211575&req=5

f1: 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.
Mentions: As shown in Fig. 1, the entire cloning procedure involves the following 4 steps.

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