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Imaging Cells in the Developing Nervous System with Retrovirus Expressing Modified Green Fluorescent Protein

Okada, Ami and Lansford, Rusty and Weimann, James M. and Fraser, Scott E. and McConnell, Susan K. (1999) Imaging Cells in the Developing Nervous System with Retrovirus Expressing Modified Green Fluorescent Protein. Experimental Neurology, 156 (2). pp. 394-406. ISSN 0014-4886. https://resolver.caltech.edu/CaltechAUTHORS:20160323-090414841

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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 β-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°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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1006/exnr.1999.7033DOIArticle
http://www.sciencedirect.com/science/article/pii/S0014488699970334PublisherArticle
ORCID:
AuthorORCID
Lansford, Rusty0000-0002-2159-3699
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 1999 by Academic Press. Received October 26, 1998; accepted November 30, 1998. Available online 1 April 2002. We thank Chris Kaznowski for technical assistance, Allison Hall for preparation of ferret brain slices, and Catherine Carswell Crumpton for performing the FACS analysis. This work was supported by ACS PF4263 to A.O., NIH EY08411 and NS12151 to S.K.M., NIH MH49176 to S.E.F., and a Caltech Biology Fellowship to R.D.L.
Funders:
Funding AgencyGrant Number
American Cancer SocietyPF4263
NIHEY08411
NIHNS12151
NIHMH49176
Caltech Division of BiologyUNSPECIFIED
Subject Keywords:GFP; retrovirus; vesicular stomatitis virus G protein; neural development; imaging; cerebral cortex; cell lineage
Issue or Number:2
Record Number:CaltechAUTHORS:20160323-090414841
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160323-090414841
Official Citation:Ami Okada, Rusty Lansford, James M. Weimann, Scott E. Fraser, Susan K. McConnell, Imaging Cells in the Developing Nervous System with Retrovirus Expressing Modified Green Fluorescent Protein, Experimental Neurology, Volume 156, Issue 2, April 1999, Pages 394-406, ISSN 0014-4886, http://dx.doi.org/10.1006/exnr.1999.7033. (http://www.sciencedirect.com/science/article/pii/S0014488699970334)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:65608
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:23 Mar 2016 17:21
Last Modified:03 Oct 2019 09:48

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