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Experimental RNAi

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...

RNA Interference

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...

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Related Experiment Video

Updated: Jul 13, 2026

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

Published on: May 24, 2022

pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells.

Soizik Berlivet¹, Virginie Guiraud, Martin Houlard

¹Commissariat a l'Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Epigenetic Regulation and Cancer Group, Cif-sur-Yvette, France.

|July 7, 2007

Summary

We developed pHYPER, an improved H1 shRNA vector for enhanced RNA interference in mouse embryonic stem cells. This new vector significantly increases shRNA expression for efficient loss-of-function studies.

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Last Updated: Jul 13, 2026

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Area of Science:

Molecular Biology
Gene Regulation
Stem Cell Biology

Background:

RNA interference (RNAi) is crucial for generating loss-of-function phenotypes.
Plasmid vectors using RNA polymerase III promoters express short hairpin RNAs (shRNAs) in mammalian cells.
Optimizing shRNA vector efficiency in embryonic stem (ES) cells is essential for research.

Purpose of the Study:

To construct and evaluate novel H1 promoter-based shRNA vectors for improved efficiency in mouse ES cells.
To compare the performance of a new vector, pHYPER, against a standard vector (pSUPER).
To provide an optimized vector for both transient and stable gene silencing applications in ES cells.

Main Methods:

Construction of a new H1 shRNA vector (pHYPER) using a 2.5-kb mouse genomic fragment containing the H1 promoter.
Comparative analysis of pHYPER and a basic 0.2-kb H1 promoter vector.
Assessment of shRNA expression levels using epifluorescence and fluorescent-activated cell sorting (FACS) after genomic integration in mouse ES cells.

Main Results:

The pHYPER vector directs significantly higher amounts of shRNA synthesis compared to the basic 0.2-kb H1 promoter vector.
pHYPER demonstrated a 4-fold increase in activity over the basic H1 vector in integrated mouse ES cells.
The enhanced expression facilitates more efficient gene silencing in ES cells.

Conclusions:

pHYPER is a superior H1 shRNA vector for gene silencing in mouse embryonic stem cells.
This optimized vector enhances RNA interference efficiency for loss-of-function studies.
pHYPER is suitable for both transient transfections and the generation of stable ES cell lines.