One-oligonucleotide method for constructing vectors for RNA interference1

Flores-Jasso, Carlos Fabián; Velázquez-Quesada, Inés; Landa-Solís, Carlos; Gutierrez, Andres; Vaca, Luis

doi:10.1111/j.1745-7254.2005.00230.x

Cited by 2 publications

(1 citation statement)

References 30 publications

(38 reference statements)

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“…For efficient and lower-cost shRNA construction, several routes was reported previously including the one-oligonucleotide method combined with PCR [14] or the four short oligonucleotides based strategy [15]. The most significant advantage to the method described in this study is that only single long (without PCR) or two short oligonucleotides were required for cloning shRNAs.…”

Section: Discussionmentioning

confidence: 99%

A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

et al. 2013

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BackgroundRNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive.ResultsIn this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a>95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo.ConclusionThis novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

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Section: Discussionmentioning

confidence: 99%

A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

et al. 2013

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Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA

Zare¹,

Sharifzadeh²,

Behzad-Behbahani³

et al. 2021

AJMB

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Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.

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One-oligonucleotide method for constructing vectors for RNA interference1

Cited by 2 publications

References 30 publications

A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA

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