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Molecular Genetics and Genomics

, Volume 287, Issue 4, pp 313–324 | Cite as

Triple mammalian/yeast/bacterial shuttle vectors for single and combined Lentivirus- and Sindbis virus-mediated infections of neurons

  • Lidia Bakota
  • Roland Brandt
  • Jürgen J. Heinisch
Original Paper

Abstract

Today, a large variety of viral vectors is available for ectopic gene expression in mammalian cell cultures or in vivo. Among them, infection with Sindbis virus- or Lentivirus-derived constructs is often used to address biological questions or for applications in neuronal therapies. However, cloning of genes of interest is time consuming, since it relies on restriction and ligation, frequently of PCR-generated DNA fragments with suitable restriction sites introduced by the primers employed. We here take advantage of the unusually high capacity for homologous recombination in Saccharomyces cerevisiae to circumvent this problem, and introduce a new set of triple shuttle vectors, which can be shuffled between E. coli, yeast, and mammalian cells. The system allows the introduction of genes of interest largely independent of the target site in the vectors. It also allows the removal of the yeast selection marker by Cre-recombinase directed recombination in E. coli, if vector size limits transfection efficiency in the mammalian cells. We demonstrate the expression of genes encoding fluorescent proteins (EGFP and mCherry) both separately and in combination, using two different viral systems in mammalian cell lines, primary neurons and organotypic slices.

Keywords

Saccharomyces cerevisiae In vivo recombination Viral vectors Fluorescent protein fusions 

Notes

Acknowledgments

We thank Andrea Murra for excellent technical assistance. We are also indebted to Sondra Schlesinger (Washington, MO), Knut Jahreis (Osnabrück, Germany), Michael Knop (Heidelberg, Germany), Pavel Osten (Chicago, IL) and Roel Nusse (Stanford, CA) for providing vectors and plasmids. Funds have been provided by the Deutsche Forschungsgemeinschaft (SFB 944) to R.B. and J.J.H., and (DFG BR1192/11-2) to R.B.

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Lidia Bakota
    • 1
  • Roland Brandt
    • 1
  • Jürgen J. Heinisch
    • 2
  1. 1.Department of Neurobiology, Faculty of Biology/ChemistryUniversity of OsnabrückOsnabrückGermany
  2. 2.Department of Genetics, Faculty of Biology/ChemistryUniversity of OsnabrückOsnabrückGermany

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