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

Autor(en): Bakota, Lidia
Brandt, Roland 
Heinisch, Juergen J.
Stichwörter: AMYLOID-BETA; Biochemistry & Molecular Biology; CORTICAL-NEURONS; Fluorescent protein fusions; GENE DELIVERY; Genetics & Heredity; HOMOLOGOUS RECOMBINATION; In vivo recombination; IN-VIVO CLONING; KLUYVEROMYCES-LACTIS; MANIPULATIONS; Saccharomyces cerevisiae; TAU; Viral vectors
Erscheinungsdatum: 2012
Volumen: 287
Ausgabe: 4
Startseite: 313
Seitenende: 324
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.
ISSN: 16174615
DOI: 10.1007/s00438-012-0680-1

Show full item record

Page view(s)

Last Week
Last month
checked on Mar 3, 2024

Google ScholarTM