In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Overview

This protocol outlines a versatile method for conditional control of endogenous gene expression in live animals or cells using enhanced lac repressor and tet activator systems. This approach enables researchers to investigate gene function at various expression levels and in a spatio-temporal manner.

Key Study Components

Area of Science

• Gene expression regulation
• Transgenic models
• Functional genomics

Background

• Existing methods for gene expression control have limitations.
• Conditional control allows for reversible manipulation of gene expression.
• This method can be applied to study disease-related genes.
• Utilizes IPTG and doxycycline for gene repression and activation, respectively.

Purpose of Study

• To develop a robust system for controlling endogenous gene expression.
• To facilitate the study of gene function in a dynamic context.
• To enable testing of phenotype reversibility in genetic studies.

Methods Used

• Engineering target gene introns with lac operators for repression.
• Using rtTA-M2 activators for upregulation of target genes.
• Combining lac repressor and tat activator systems for dual control.
• Employing qRT-PCR and immunostaining for expression analysis.

Main Results

• Demonstrated effective repression of DNMT1 expression to 15% of unregulated levels.
• Repression was reversible in a dose-dependent manner with IPTG treatment.
• Achieved over 90% repression of mKate2 expression using intron-based approaches.
• Validated results through confocal imaging and qRT-PCR analysis.

Conclusions

• The method provides a powerful tool for gene expression studies.
• Offers insights into gene function and disease mechanisms.
• Facilitates the exploration of gene regulation in live models.

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