Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein

Exp Neurol. 1999 Apr;156(2):394-406. doi: 10.1006/exnr.1999.7033.

Abstract

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / genetics
  • Animals
  • Axons / ultrastructure
  • Cell Lineage
  • Cerebral Cortex / cytology
  • Cerebral Cortex / embryology*
  • Cytomegalovirus / genetics
  • Dendrites / ultrastructure
  • Enhancer Elements, Genetic
  • GAP-43 Protein / genetics
  • Genes, Reporter*
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • Membrane Proteins / analysis
  • Membrane Proteins / genetics
  • Microscopy, Video
  • Moloney murine leukemia virus / genetics*
  • Neural Crest / cytology*
  • Neurons / cytology
  • Promoter Regions, Genetic
  • Rats
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / biosynthesis
  • Terminal Repeat Sequences
  • Vesicular stomatitis Indiana virus / physiology*

Substances

  • Actins
  • GAP-43 Protein
  • Luminescent Proteins
  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins