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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...

siRNA - Small Interfering RNAs

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...

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Related Experiment Video

Updated: Jul 20, 2026

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Published on: December 12, 2017

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene

Kum-Joo Shin¹, Estelle A Wall, Joelle R Zavzavadjian

¹Alliance for Cell Signaling Molecular Biology Laboratory, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

Proceedings of the National Academy of Sciences of the United States of America

|September 2, 2006

Summary

Researchers developed a novel lentiviral vector system (pSLIK) for inducible gene knockdown and simultaneous transgene expression. This tool enables precise control over gene function studies in mammalian cells, advancing genome annotation.

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Area of Science:

Molecular Biology
Genomics
Gene Regulation

Background:

RNA interference (RNAi) is crucial for mammalian genome functional annotation.
Regulated manipulation of endogenous gene expression alongside exogenous transgene reexpression is key to realizing RNAi's full potential.
Existing methods require complex approaches for coordinated gene knockdown and reexpression.

Purpose of the Study:

To develop a versatile lentiviral vector platform for tetracycline-regulated gene knockdown.
To enable coordinated reexpression of transgenes concurrently with endogenous gene knockdown.
To demonstrate the utility of this system in studying gene function in mammalian cells.

Main Methods:

Development of the pSLIK (single lentivector for inducible knockdown) system, a single lentiviral vector for tetracycline-regulated microRNA-like short hairpin RNA expression.
Application of the pSLIK platform in mouse embryonic fibroblasts to conditionally deplete Galpha12 and Galpha13 heterotrimeric G proteins.
Utilized RAW264.7 macrophages to assess the impact of Gbeta2 knockdown on calcium signaling.
Incorporated a GFP transgene upstream of the short hairpin RNA for simultaneous gene knockdown and reexpression.

Main Results:

Demonstrated Galpha13 dependence of serum response element-mediated transcription using the pSLIK system.
Observed reduced Ca(2+) response to C5a in RAW264.7 macrophages upon regulated knockdown of Gbeta2.
Successfully achieved simultaneous tetracycline-dependent target gene knockdown and heterologous mRNA reexpression (GFP) using the pSLIK system.

Conclusions:

The pSLIK system provides a robust platform for inducible knockdown of endogenous genes in various mammalian cell types.
This system facilitates the study of gene function by enabling coordinated knockdown and reexpression, enhancing experimental applicability.
The pSLIK technology represents a significant advancement for functional genomics and mammalian genome annotation.