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Related Concept Videos

Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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An EYFP insertion mutant containing a modified lox sequence for potential use as a recombination indicator.

Akiko Mizutani1, Masato Ohtsuka, Minoru Kimura

  • 1Depertment of Molecular Life Sciences, Division of Basic Molecular Science and Molecular Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.

Nucleic Acids Symposium Series (2004)
|December 8, 2006
PubMed
Summary

Researchers developed EYFP157, a novel fluorescent protein variant, to improve screening for intermolecular Cre-loxP recombination events in genetic engineering. This tool enhances the detection of rare recombination products in E. coli and mouse cells.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The Cre-loxP system is a widely used genetic engineering tool.
  • Intermolecular recombination is less frequent than intramolecular recombination, posing screening challenges.
  • Efficient screening methods are crucial for isolating intermolecular Cre-loxP recombination products.

Purpose of the Study:

  • To construct a novel EYFP variant (EYFP157) for enhanced screening of intermolecular Cre-loxP recombination.
  • To assess the utility of EYFP157 as a recombination indicator in E. coli and mouse cells.

Main Methods:

  • Construction of EYFP157 containing a non-cleavable mutant loxP sequence.
  • Expression and fluorescence detection of EYFP157 in E. coli and mouse cells at different temperatures.
  • Comparison of EYFP157 fluorescence with wild-type EYFP.

Main Results:

  • EYFP157 exhibited detectable fluorescence in E. coli and mouse cells at 37°C, albeit at 1-3% of wild-type EYFP levels.
  • At room temperature, EYFP157 fluorescence in E. coli cells approached that of wild-type EYFP.
  • The mutant loxP sequence within EYFP157 was resistant to Cre-mediated cleavage.

Conclusions:

  • EYFP157 is a promising tool for screening intermolecular Cre-loxP recombination.
  • The temperature-dependent fluorescence properties of EYFP157 offer versatile applications.
  • This variant facilitates the isolation of rare intermolecular recombination products in genetic engineering.