A dicistronic retroviral vector and culture model for analysis of neuron-Schwann cell interactions.
A dicistronic retroviral gene delivery system and tissue culture model has been developed for studies of neuron-Schwann cell interactions at the single cell level. The dicistronic retroviral vector contains a multiple cloning site followed by the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES) and a green fluorescent protein gene. This design allows for 5'-cap dependent translation of any gene of interest and 5'-cap independent translation of green fluorescent protein (GFP) from a single dicistronic RNA. The culture model consists of dorsal root ganglia (DRG) explants grown in defined medium. Under these conditions the Schwann cell population is selectively expanded and infected by the retroviral vector, allowing for rapid transfer of genes of interest selectively to a large percentage of Schwann cells in coculture with neurons. Infected cells are subsequently identified in living cultures by their expression of GFP. Infected (GFP expressing) Schwann cells in contact with neurites continued to exhibit: (1) increased mitotic activity, (2) increased sensitivity to elevate intracellular calcium in response to extracellular application of ATP, and (3) myelination. This viral construct has the added advantage that it allows identification of cells expressing transgenes among a heterogeneous population by fluorescence microscopy, FACS, or flow cytometry.
Howe
D G
DG
Neurobiology, The University of North Carolina at Chapel Hill, 27599, USA.
McCarthy
K D
KD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology / Q000821:virology; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008938:Mitosis / Q000502:physiology; D008979:Moloney murine leukemia virus; D009185:Myelin Proteins / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012192:Retroviridae Infections; D012270:Ribosomes / Q000502:physiology; D012583:Schwann Cells / Q000737:chemistry / Q000166:cytology* / Q000502:physiology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009185:Myelin Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D002118:Calcium