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- Publisher
- Springer Journals
- Copyright
- Copyright © 2012 by Springer-Verlag
- Subject
- Life Sciences; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics & Genomics; Cell Biology; Biochemistry, general
- ISSN
- 1617-4615
- eISSN
- 1617-4623
- DOI
- 10.1007/s00438-012-0680-1
- pmid
- 22327349
- Publisher site
- See Article on Publisher Site
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.
Journal
Molecular Genetics and Genomics – Springer Journals
Published: Feb 12, 2012