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Related Experiment Videos

Functional cloning using pFB retroviral cDNA expression libraries.

Katherine A Felts1, Keith Chen, Kim Zaharee

  • 1Applied Molecular Evolution, San Diego, California 92121, USA.

Molecular Biotechnology
|October 2, 2002
PubMed
Summary

Researchers developed retroviral complementary DNA (cDNA) expression libraries for efficient gene function screening in mitotic cells. This method allows precise control over gene copy number, enabling the isolation of key genes like oncogenes and cell-surface receptors.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Virology

Background:

  • Retroviral cDNA expression libraries offer a powerful tool for introducing complex genetic material into cells.
  • Efficiently screening genes based on function is crucial for understanding cellular processes and disease mechanisms.

Purpose of the Study:

  • To describe the development and application of retroviral cDNA expression libraries for gene discovery.
  • To demonstrate the isolation of specific genes, including oncogenes and cell-surface receptors, using this technology.

Main Methods:

  • Construction of a high-titer retroviral vector (pFB) containing a complementary DNA (cDNA) library from human Burkitt's lymphoma (Daudi) cells.
  • Introduction of the retroviral cDNA library into mitotic cells.
  • Control of complementary DNA (cDNA) copy number per cell via multiplicity of infection.

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Main Results:

  • Generation of cell populations where over 90% of infected cells contained one to three complementary DNA (cDNA) copies.
  • Successful isolation of two known oncogenes from the library.
  • Successful isolation of one cell-surface receptor from the library.

Conclusions:

  • Retroviral cDNA expression libraries provide an efficient method for introducing complex genetic material into diverse cell types for functional screening.
  • This approach allows for precise control over gene expression levels, facilitating the identification of functionally significant genes.