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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...

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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Updated: Jun 27, 2026

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Published on: March 29, 2019

Rapid, controlled and intensive lentiviral vector-based RNAi.

Manuel Llano¹, Natassia Gaznick, Eric M Poeschla

¹Molecular Medicine Program, Molecular Medicine Program, Rochester, MN, USA.

Methods in Molecular Biology (Clifton, N.J.)

|November 21, 2008

Summary

This study presents an optimized RNA interference (RNAi) method using lentiviral vectors for deep gene knockdown. This technique enables effective gene function studies in human cells, including T cells and macrophages, with rapid data acquisition.

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Last Updated: Jun 27, 2026

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Published on: March 29, 2019

Lentivirus Production

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Published on: October 2, 2009

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

Area of Science:

Molecular Biology
Gene Regulation
Virology

Background:

RNA interference (RNAi) is crucial for gene function studies but can be limited by insufficient knockdown and control rigor.
Lentiviral vectors offer efficient gene transfer, enhancing RNAi efficacy.
Optimizing short hairpin RNA (shRNA) expression with lentiviral delivery is key for robust gene silencing.

Purpose of the Study:

To review and detail an optimized RNAi method combining pol III promoters and lentiviral vectors.
To demonstrate effective and convincing gene knockdown in human cell lines and primary cells.
To highlight practical methods for rapid gene function analysis.

Main Methods:

Utilizing pol III promoters for short hairpin RNA (shRNA) expression.
Employing lentiviral vectors for efficient gene transfer and stable integration.
Implementing one-step vector construction for streamlined experimental design.
Performing RNAi-resistant gene re-expression for phenotype rescue validation.

Main Results:

Achieved deep and stable mRNA reductions in T cell lines.
Demonstrated successful application in HIV challenge studies.
Validated the method's effectiveness in primary T cells and macrophages.
Showcased rapid experimental turnaround from target identification to data readout (weeks).

Conclusions:

Optimized RNAi using lentiviral vectors provides a powerful and accessible tool for gene function studies.
The described methods ensure rigorous controls and effective gene knockdown.
This approach accelerates biological discovery in various human cell types.