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Related Experiment Video

Updated: Dec 20, 2025

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System
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Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro.

Tuyana Malankhanova1, Michael Sorokin1, Sergey Medvedev1

  • 1Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

Current Protocols in Human Genetics
|May 30, 2020
PubMed
Summary
This summary is machine-generated.

Researchers developed a new protocol using CRISPR/Cas9 to create Huntington's disease cell models. This method precisely introduces expanded CAG repeats into the HTT gene, enabling study of disease mechanisms.

Keywords:
Huntington's diseasegenome editingisogenic cell linespolyglutamine diseases

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Area of Science:

  • Genetics
  • Molecular Biology
  • Neuroscience

Background:

  • Neurodegenerative diseases like Huntington's disease (HD) present challenges due to late manifestation and limited material.
  • Cellular models are crucial for understanding pathogenesis, and genome editing facilitates the creation of isogenic models for hereditary diseases.

Purpose of the Study:

  • To outline a protocol for generating isogenic cell models of Huntington's disease by introducing expanded CAG repeat tracts into the HTT gene.
  • To enable modeling of HD at various severity levels and provide a framework for other trinucleotide repeat expansion disorders.

Main Methods:

  • Utilizing CRISPR/Cas9-mediated homologous recombination.
  • Designing a specialized donor plasmid with an expanded CAG tract and long homology arms.
  • Implementing protocols for plasmid assembly, cell transfection, GFP-positive cell sorting, and PCR screening for mutant HTT expression.

Main Results:

  • Successful introduction of expanded CAG repeat tracts into the HTT gene.
  • Generation of isogenic cell models reflecting different Huntington's disease severity levels.
  • Troubleshooting solutions for challenges associated with cloning repeated and GC-rich sequences.

Conclusions:

  • The developed protocol offers a robust method for creating Huntington's disease cell models.
  • This approach is adaptable for modeling other trinucleotide repeat expansion diseases.
  • The protocol addresses common technical hurdles in manipulating repetitive DNA sequences.