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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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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Published on: January 8, 2015

Recombination-ready Sindbis replicon expression vectors for transgene expression.

Brian J Geiss1, Lisa H Shimonkevitz, Cherilyn I Sackal

  • 1Arthropod-Borne and Infectious Diseases Laboratory, Department of Molecular Biology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Brian.Geiss@colostate.edu

Virology Journal
|October 30, 2007
PubMed
Summary

This study introduces a new system for creating Sindbis virus replicons, significantly improving efficiency and speed for gene function studies. The method ensures correct insert orientation, simplifying the use of alphavirus replicons.

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Recombineering Homologous Recombination Constructs in Drosophila
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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Recombineering Homologous Recombination Constructs in Drosophila
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Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Area of Science:

  • Molecular Biology
  • Virology
  • Gene Expression Systems

Background:

  • Sindbis viruses are valuable tools for studying gene function.
  • Traditional alphavirus replicon construction is inefficient and time-consuming.
  • Existing methods face challenges with insert orientation and restriction sites.

Purpose of the Study:

  • To develop a rapid and specific system for constructing recombinant Sindbis replicons.
  • To improve the efficiency of creating expression plasmids for alphavirus replicons.
  • To facilitate the study of gene function using alphavirus systems.

Main Methods:

  • Utilized lambda phage recombination technology for plasmid construction.
  • Developed a system for rapid and specific insertion of genes into replicon plasmids.
  • Produced pseudo-infectious viral particles for cell culture experiments.

Main Results:

  • Achieved nearly 100% recombination efficiency with correct insert orientation.
  • Successfully produced and packaged replicons into pseudo-infectious viral particles.
  • Demonstrated high-level transgene expression in insect and mammalian cells.
  • Enabled easy isolation and re-cloning of inserts from replicating RNA for analysis.

Conclusions:

  • Replication-ready expression plasmids streamline alphavirus replicon production.
  • The new system significantly enhances the speed and ease of using alphavirus replicons.
  • This advancement improves the utility of alphavirus replicons for gene function research.