title: Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.
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.
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D001369:Axons / Q000648:ultrastructure; D019070:Cell Lineage; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology*; D003587:Cytomegalovirus / Q000235:genetics; D003712:Dendrites / Q000648:ultrastructure; D004742:Enhancer Elements, Genetic; D019922:GAP-43 Protein / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D018715:Microscopy, Video; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009432:Neural Crest / Q000166:cytology*; D009474:Neurons / Q000166:cytology; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D020079:Terminal Repeat Sequences; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology*