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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...

In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

MicroRNAs

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...

MicroRNAs

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...

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Related Experiment Video

Updated: Jun 10, 2026

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

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Published on: March 14, 2018

Efficient gene knockdowns in mouse embryonic stem cells using microRNA-based shRNAs.

Jianlong Wang¹

¹Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY, USA.

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

|August 6, 2010

Summary

This study details protocols for RNA interference (RNAi) gene knockdown in mouse embryonic stem cells (ESCs). It focuses on using microRNA-embedded short-hairpin RNA (shRNA(mir)) for functional genetic studies and cell therapy applications.

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CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Published on: August 24, 2022

Area of Science:

Molecular Biology
Genetics
Stem Cell Biology

Background:

RNA interference (RNAi) is a key technology for gene knockdown in eukaryotic systems.
Embryonic stem cells (ESCs) are crucial for understanding self-renewal, pluripotency, and cell-type specification for therapies.
Effective RNAi in ESCs requires robust gene knockdown methods.

Purpose of the Study:

To provide detailed protocols for RNA interference (RNAi) gene knockdown in mouse ESCs.
To enable functional genetic loss-of-function studies in ESCs.
To facilitate research into ESC self-renewal, pluripotency, and cell differentiation for therapeutic applications.

Main Methods:

Utilizing microRNA-embedded short-hairpin RNA (shRNA(mir)) cassettes for potent RNAi response.
Employing retrovirus infection for efficient constitutive gene knockdown.
Implementing loxP site-directed recombination for inducible gene knockdown.

Main Results:

Established protocols for shRNA(mir)-mediated RNAi in mouse ESCs.
Demonstrated efficient constitutive and inducible gene knockdown strategies.
Provided a framework for dissecting gene function in ESCs.

Conclusions:

shRNA(mir)-mediated RNAi is a powerful tool for genetic studies in mouse ESCs.
Retrovirus infection and loxP recombination offer versatile methods for gene knockdown.
These protocols support research in stem cell biology and cell-replacement therapies.