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

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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.

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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

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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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Authors and Affiliations

  1. Institute of Anatomy, Anatomy and Cell Biology, University of Bonn, Nussallee 10, 53125, Bonn, Germany

    Sujeet Kumar & Stephan L. Baader

  2. Institute of Pharmacology and Toxicology, Biomedical Center, University of Bonn, Bonn, Germany

    Katrin Zimmermann & Alexander Pfeifer

  3. International Graduate School Biotech Pharma, University of Bonn, Bonn, Germany

    Katrin Zimmermann & Alexander Pfeifer

  4. Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Hiroyuki Hioki

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  1. Sujeet Kumar
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  2. Katrin Zimmermann
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  3. Hiroyuki Hioki
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  4. Alexander Pfeifer
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  5. Stephan L. Baader
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Correspondence to Stephan L. Baader.

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Kumar, S., Zimmermann, K., Hioki, H. et al. Efficient and graded gene expression in glia and neurons of primary cerebellar cultures transduced by lentiviral vectors. Histochem Cell Biol 143, 109–121 (2015). https://doi.org/10.1007/s00418-014-1260-8

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