Document Detail


Sorting human beta-cells consequent to targeted expression of green fluorescent protein.
MedLine Citation:
PMID:  9836534     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.
Authors:
K Meyer; J C Irminger; L G Moss; L M de Vargas; J Oberholzer; D Bosco; P Morel; P A Halban
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Diabetes     Volume:  47     ISSN:  0012-1797     ISO Abbreviation:  Diabetes     Publication Date:  1998 Dec 
Date Detail:
Created Date:  1998-12-21     Completed Date:  1998-12-21     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0372763     Medline TA:  Diabetes     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1974-7     Citation Subset:  AIM; IM    
Affiliation:
Laboratoires de Recherche Louis-Jeantet, University of Geneva Medical Center, Switzerland.
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MeSH Terms
Descriptor/Qualifier:
Adenoviridae / genetics
Animals
Cell Separation
Flow Cytometry
Gene Expression Regulation
Green Fluorescent Proteins
Humans
Insulin / genetics
Islets of Langerhans / cytology*,  metabolism*
Luminescent Proteins / genetics*
Promoter Regions, Genetic / genetics
Rats
Recombinant Fusion Proteins / genetics
Grant Support
ID/Acronym/Agency:
DK-51111/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 0/Recombinant Fusion Proteins; 11061-68-0/Insulin; 147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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