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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.

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...

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Related Experiment Video

Updated: Jul 18, 2026

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Published on: November 23, 2017

Rapid conditional knock-down-knock-in system for mammalian cells.

Michael Hölzel¹, Michaela Rohrmoser, Mathias Orban

¹Institute of Clinical Molecular Biology and Tumour Genetics, GSF Research Center, Marchioninistrasse 25, 81377 Munich, Germany. hoelzel@gsf.de

Nucleic Acids Research

|December 16, 2006

Summary

This study introduces a rapid knock-down and knock-in system for mammalian cells, enabling precise gene function analysis by distinguishing off-target effects. The system effectively rescues gene function loss, allowing detailed study of protein mutants.

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Area of Science:

Molecular Biology
Cell Biology
Genetics

Background:

RNA interference (RNAi) is crucial for gene function analysis in mammalian cells.
Off-target effects and compound phenotypes can complicate RNAi studies.
Replacing endogenous proteins with ectopic forms helps isolate specific gene functions.

Purpose of the Study:

To develop a rapid knock-down and knock-in system for mammalian cells.
To enable the analysis of essential genes and protein mutants.
To exclude off-target effects in gene function studies.

Main Methods:

Development of a dual episomal vector system for simultaneous knock-down and knock-in.
Doxycycline-dependent gene reconstitution and depletion using artificial miRNA-embedded siRNA.
Generation of stable polyclonal cell lines within two weeks.
Functional analysis of 17 WDR12 mutants in depleted cells.

Main Results:

The system allows for doxycycline-dependent knock-down of endogenous mRNA while enabling expression of ectopic constructs.
Stable cell lines were generated rapidly, facilitating timely experiments.
Wild-type and six WDR12 mutants rescued loss-of-function phenotypes, while others did not.
The system successfully distinguished functional roles of different protein mutants.

Conclusions:

The developed system effectively excludes off-target effects in gene function studies.
It is suitable for the functional analysis of protein mutants in depleted cellular environments.
This method provides a robust tool for dissecting gene function and protein complex roles.