Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Identification of frame-shift intermediate mutant cells.

Christoph Gasche1, Christina L Chang, Loki Natarajan

  • 1Department of Medicine and Cancer Center, University of California at San Diego, La Jolla, CA 92093-0688, USA. christoph.gasche@akh-wien.ac.at

Proceedings of the National Academy of Sciences of the United States of America
|February 13, 2003
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

The Gut Microbiome and Colorectal Cancer: From Association to Causation.

Cancer biome and targeted therapy·2026
Same author

GLP-1 receptor agonist targets an AMPKα1-HIF1α-PFKFB3 metabolic vulnerability and enhances Lenvatinib response in hepatocellular carcinoma.

NPJ precision oncology·2026
Same author

EXONERATE-TRaCE: A Liquid Biopsy for Tracking Response to Anti-Epidermal Growth Factor Receptor-Based Therapy in Metastatic Colorectal Cancer.

JCO precision oncology·2026
Same author

Publisher Correction: Integrated Liquid Biopsy and Tumor Tissue Genomic Profiling of Appendiceal Cancer: cfDNA Burden, Mutation Landscapes, and Clinical Outcomes.

Annals of surgical oncology·2026
Same author

Liquid biopsy biomarkers for early detection of gastrointestinal cancers: Current landscape and emerging technologies.

Clinical and translational medicine·2026
Same author

Andrographis exerts antitumor effects and enhances 5‑FU efficacy via the alteration of ferroptosis‑related genes in esophageal squamous cell carcinoma.

Oncology reports·2026
Same journal

Chemotactic self-organization captures the dynamics of mammalian hair follicle patterning.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same journal

Tomographic imaging of superconducting order using particle-hole interference.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same journal

Inhibitory potential of autologous neutralizing antibodies sets quantitative limits on the rebound-competent HIV-1 reservoir.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same journal

Inferring epidemiological parameters under an infectious phylogeography model with visitor dynamics.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same journal

Analytical modeling for suction cup designs for skin-interfaced wearable devices.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same journal

Improving cell-free metabolism through direct integration of artificial respiratory chains.

Proceedings of the National Academy of Sciences of the United States of America·2026
See all related articles

Frame-shift mutations arise from polymerase errors and failed mismatch repair. This study identifies intermediate mutant cells, revealing that mismatch repair proficiency does not affect their generation.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • Microsatellite instability, a hallmark of certain cancers, arises from DNA polymerase errors during replication.
  • Post-replicational mismatch repair (MMR) systems are crucial for correcting these errors, preventing mutations.
  • Understanding the kinetics of mutation generation, including intermediate states, is vital for cancer biology.

Purpose of the Study:

  • To develop a cell-culture system for identifying and characterizing intermediate mutant cells in frame-shift mutation processes.
  • To investigate the role of mismatch repair proficiency in the generation and accumulation of these intermediate mutants.
  • To quantify mutation rates and intermediate cell proportions in MMR-deficient and MMR-proficient colorectal cancer cells.

Main Methods:

Related Experiment Videos

  • Constructed a plasmid with a poly(dC-dA).poly(dG-dT) microsatellite inserted into the enhanced Green Fluorescent Protein (EGFP) gene reading frame.
  • Introduced the plasmid into MMR-deficient (HCT116, hMLH1-mutated) and MMR-proficient (HCT116+chr3, hMLH1 wild type) human colorectal cancer cells.
  • Utilized flow cytometry to detect EGFP-expressing cells and quantify distinct fluorescent populations (M1 and M2) over time.

Main Results:

  • Identified two fluorescent cell populations: M1 (dim) and M2 (bright), with M2 cells accumulating over time in MMR-deficient cells.
  • Single M2 cells in HCT116 clones yielded colonies with a 2-bp deletion at the microsatellite, indicating restored reading frame.
  • A significant proportion of M1 cells in HCT116 clones exhibited mixed fluorescence, suggesting they represent heteroduplex intermediates.

Conclusions:

  • The generation of intermediate frame-shift mutants (M1 cells) is independent of mismatch repair proficiency.
  • MMR-deficient cells exhibit a significantly higher mutation rate compared to MMR-proficient cells.
  • The developed cell-culture system effectively distinguishes between intermediate and final mutant states in microsatellite mutation.