Histochemistry and Cell Biology

, Volume 143, Issue 1, pp 109–121 | Cite as

Efficient and graded gene expression in glia and neurons of primary cerebellar cultures transduced by lentiviral vectors

  • Sujeet Kumar
  • Katrin Zimmermann
  • Hiroyuki Hioki
  • Alexander Pfeifer
  • Stephan L. Baader
Original Paper

Abstract

Lentiviral vectors are valuable tools to express genes of interest in living animals and stem cell cultures. The use of promoters in lentiviral constructs has been successfully used to drive gene expression in particular cell types including neurons and glia of the central nervous system in vivo. However, their suitability in cell culture is less well documented. In this paper, we describe lentiviral vectors containing neuronal promoters of the murine stem cell virus, of the synapsin 1 gene, the tubulin alpha 1 gene, and the calmodulin kinase II gene, and the glial promoter of the glial fibrillary acidic protein gene to drive reporter gene expression in primary dissociated cerebellar cell cultures and in slice cultures. While the glial promoter was highly specific for glia, the neuronal promoters were active in neurons and glia of dissociated cultures to a comparable extent. In slice cultures, neuronal and glial promoters demonstrated higher, but not absolute selectivity for particular cell types. In addition, the promoters allowed for an efficient and graded expression of genes in dissociated cultures. By using selected combinations of vectors, it was also possible to drive the expression of two genes in one cell type with high efficiency. A gene of interest in combination with a reporter gene can thus be expressed in a graded manner to reveal gene function in a rather short time and in a complex cellular environment.

Keywords

Cerebellum Lentiviral vector transduction Slice culture Primary culture Promoter Transfection 

Abbreviations

MBP

Myelin basic protein

GFAP

Glial fibrillary acidic protein

MSCV

Murine stem cell virus

CMV

Cytomegalovirus

Syn1

Synapsin 1

Ta1

Tubulin alpha 1

CaMKII

Calmodulin kinase II

CAG

Chicken beta actin

BSA

Bovine serum albumin

GFP/RFP

Green/red fluorescent protein

MAP

Microtubule-associated protein

Notes

Acknowledgments

All authors gave their informed consent prior to their inclusion in the study. We very much appreciate the excellent technical help of Stefanie Ramrath and Helma Langmann. We also are grateful to the help of Daniela Krauss and Franz Neuhalfen for providing timed pregnant mice. The MSCV promoter was kindly provided by Dr. Neumann (Institute of Reconstructive Neurobiology, University of Bonn) and the GFAP promoter by Dr. Sock (Institute of Biochemistry, University of Erlangen).

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Sujeet Kumar
    • 1
  • Katrin Zimmermann
    • 2
    • 3
  • Hiroyuki Hioki
    • 4
  • Alexander Pfeifer
    • 2
    • 3
  • Stephan L. Baader
    • 1
  1. 1.Institute of Anatomy, Anatomy and Cell BiologyUniversity of BonnBonnGermany
  2. 2.Institute of Pharmacology and Toxicology, Biomedical CenterUniversity of BonnBonnGermany
  3. 3.International Graduate School Biotech PharmaUniversity of BonnBonnGermany
  4. 4.Department of Morphological Brain Science, Graduate School of MedicineKyoto UniversityKyotoJapan

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