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Conservative Site-specific Recombination and Phase Variation02:53

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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.
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Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
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LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in mammals compared to other class I transposable elements. About 8 percent of human genomic DNA comprises LTR retrotransposons. Some of the common examples of LTR retrotransposons are Ty elements in yeast and Copia elements in Drosophila.
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Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
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Simplifying transgene locus structure through Cre-lox recombination.

Vibha Srivastava1, David W Ow

  • 1Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, USA, vibhas@uark.edu.

Methods in Molecular Biology (Clifton, N.J.)
|March 6, 2015
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Summary

Multicopy transgene silencing in plants can be overcome by using the Cre-lox system to convert multiple copies into single copies, restoring gene expression. This method aids in transforming difficult crops.

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Area of Science:

  • Plant biotechnology
  • Molecular biology
  • Genetics

Background:

  • Transgene silencing is a common issue in genetically modified plants, often linked to multiple copies of the integrated transgene.
  • Restoring transgene expression is crucial for effective genetic engineering in crops.

Purpose of the Study:

  • To describe a novel plant transformation protocol utilizing the Cre-lox system.
  • To enable the conversion of multicopy transgene loci into single-copy or low-copy states to restore gene expression.

Main Methods:

  • Designing a transformation vector incorporating lox sites.
  • Developing transgenic plant lines containing the designed vector.
  • Introducing Cre recombinase activity to induce Cre-lox recombination for locus simplification.

Main Results:

  • Successfully demonstrated the conversion of multicopy transgene loci to single- or low-copy states.
  • The Cre-lox system effectively simplified transgene loci, mitigating silencing effects.

Conclusions:

  • The Cre-lox system offers a viable strategy to address transgene silencing caused by multicopy integrations.
  • This protocol is compatible with common plant transformation methods (gene gun, Agrobacterium) and beneficial for recalcitrant crops.