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Exogenous DNA expression in eukaryotic cells following microinjection.

Ann Boyd1

  • 1Biology Department, Hood College, 401 Rosemont Ave., Frederick MD 21701, USA. boyd@hood.edu

Methods in Cell Science : an Official Journal of the Society for in Vitro Biology
|July 5, 2003
PubMed
Summary

Microinjection enables direct observation of gene expression in single eukaryotic cells, bypassing toxicity issues common in other methods. This technique allows for efficient monitoring of foreign gene product expression and intracellular reactions in real-time.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Facilitated transfer protocols for foreign material into eukaryotic cells require large cell numbers and can cause uptake toxicity.
  • Microinjection offers a direct method for introducing nucleic acids, proteins, and soluble materials into single living cells.
  • Single-cell analysis is crucial for understanding cellular responses and gene expression dynamics.

Purpose of the Study:

  • To highlight the advantages of microinjection for single-cell experiments.
  • To demonstrate the efficiency and direct observation capabilities of microinjection for gene expression studies.
  • To showcase the use of fluorescent markers for real-time monitoring of gene expression.

Main Methods:

  • Microinjection of DNA, RNA, proteins, and soluble materials directly into the nucleus or cytoplasm of eukaryotic cells.

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  • Utilizing marker fluorescent proteins, such as green fluorescent protein (GFP), fused with test genes for simultaneous expression monitoring.
  • Quantifying expression efficiency by counting the percentage of injected cells exhibiting the foreign gene product.

    • Main Results:

    • Microinjection allows direct monitoring of treated cells and real-time observation of intracellular reactions and expressed products.
    • Expression of foreign genes, like SV40 T antigen, can be detected as early as four hours post-microinjection.
    • Fusion proteins, such as T-GFP, allow for non-invasive, sequential observation of cellular processes without fixation.

    Conclusions:

    • Microinjection is a superior method for single-cell gene expression studies, offering direct observation and bypassing uptake toxicity.
    • The technique allows for accurate determination of expression efficiency as a percentage of injected cells.
    • Microinjection facilitates the study of gene expression across various cell types and enables real-time monitoring of cellular responses to exogenous materials.