Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Overview

This protocol describes a method for generating cancer cell clones with a MS2 sequence tag at a single subtelomere, allowing for the visualization of TERRA transcripts in living cells.

Key Study Components

Area of Science

• Cell Biology
• Cancer Research
• Genetics

Background

• Telomeres play a crucial role in chromosome stability.
• TERRA is a noncoding RNA associated with telomeres.
• Understanding TERRA localization can provide insights into telomere function.
• Current methods lack the ability to visualize TERRA from a single telomere.

Purpose of Study

• To develop a technique for visualizing TERRA from a specific telomere.
• To investigate the localization of TERRA in living cells.
• To enhance understanding of telomere biology.

Methods Used

• Culture AGS cells to 50-60% confluence.
• Transfect cells with sgRNA Cas9 vector and MS2 cassette.
• Replace medium with Neomycin-containing medium.
• Use Tripsyn for cell detachment in a 96-well plate.

Main Results

• The method successfully visualizes TERRA transcripts in living cells.
• Demonstrated the localization of TERRA to a single telomere.
• Provided a new tool for studying telomere dynamics.
• Enabled further exploration of telomere-related questions.

Conclusions

• This protocol offers a novel approach to study TERRA.
• It enhances the understanding of telomere function in cancer biology.
• Future studies can leverage this method for deeper insights.

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