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Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides

V Alexeev1, O Igoucheva, K Yoon

  • 1Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA, USA.

Gene Therapy
|November 29, 2002
PubMed

Insights

Single-stranded oligodeoxynucleotides (ODNs) efficiently correct gene mutations and activate genes in mammalian cells. This study demonstrates simultaneous gene targeting, offering a more reproducible method than RNA-DNA oligonucleotides (RDOs).

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Oligonucleotides, including chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligodeoxynucleotides (ODNs), are investigated for their potential in chromosomal gene alteration.
  • Previous studies have shown the capability of these molecules in modifying mammalian cell genes.

Purpose of the Study:

  • To evaluate the efficacy of single-stranded oligodeoxynucleotides (ODNs) in correcting gene mutations and introducing new mutations in mammalian cells.
  • To compare the gene targeting efficiency and reproducibility of ODNs with RDOs.
  • To demonstrate the feasibility of simultaneous targeting of two distinct genes within a single cell.

Main Methods:

  • Utilized two ODNs to correct an inactivating mutation in the tyrosinase gene and introduce an activating mutation into the c-kit gene in mouse melanocytes.
  • Assessed gene targeting frequency by observing pigmentation changes resulting from tyrosinase gene correction.
  • Performed DNA sequence analysis on converted cells to confirm targeted modifications.

Main Results:

  • Achieved gene targeting frequencies for ODNs ranging from 2 x 10(-4) to 1 x 10(-3), comparable to RDOs.
  • ODNs demonstrated significantly higher reproducibility in gene correction (60% of experiments) compared to RDOs (10%).
  • Successfully demonstrated simultaneous targeting of both the tyrosinase and c-kit genes in a single cell, leading to restored melanin production and activated Kit receptor kinase.

Conclusions:

  • Single-stranded oligodeoxynucleotides (ODNs) are a viable and more reproducible tool for chromosomal gene alterations in mammalian cells compared to RDOs.
  • Simultaneous targeting of multiple genes in a single cell is feasible using ODNs.
  • A selection strategy can effectively enrich cells with desired gene alterations after targeted modification.

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