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Related Experiment Videos

Phage R4 integrase mediates site-specific integration in human cells.

E C Olivares1, R P Hollis, M P Calos

  • 1Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.

Gene
|November 15, 2001
PubMed
Summary
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The R4 integrase, a phage-derived enzyme, efficiently mediates site-specific DNA integration in human cells. This recombinase enables precise genome engineering by targeting specific attB and attP sites for DNA insertion.

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Site-specific recombinases are crucial tools for precise DNA manipulation in biotechnology and genetic engineering.
  • Phage-derived integrases offer unique properties for targeted DNA integration.
  • The R4 integrase from Streptomyces parvulus phage R4 was previously uncharacterized for its genome engineering potential.

Purpose of the Study:

  • To characterize the R4 integrase for its ability to perform site-specific DNA recombination in mammalian cells.
  • To define the minimal recognition sites (attB and attP) for the R4 integrase.
  • To evaluate the R4 integrase as a tool for genome engineering applications.

Main Methods:

  • Cloning and expression of the R4 integrase gene in mammalian cells.

Related Experiment Videos

  • Design and synthesis of compact attB and attP recognition sites.
  • Assessing recombination efficiency between attB and attP sites on extrachromosomal vectors in human cells.
  • Investigating the integration of plasmids into endogenous genomic sequences using R4 att sites.
  • Utilizing phi C31 integrase to pre-integrate R4 att sites into the human genome as targets.

    • Main Results:

    • The R4 integrase was successfully expressed and functional in human cells.
    • Efficient and precise recombination was achieved between R4 attB and attP sites on plasmids.
    • The R4 integrase mediated the integration of plasmids into endogenous genomic DNA.
    • Engineered genomic sites (R4 attB/attP) served as effective targets for incoming R4 att-bearing plasmids.

    Conclusions:

    • The R4 integrase is a functional site-specific recombinase in human cells.
    • Compact R4 att sites facilitate precise and efficient DNA integration.
    • The R4 integrase is a valuable tool for genome engineering, with potential for further development.