DUAL-FUNCTION REPORTER PROTEIN FOR ANALYSIS OF GENE-EXPRESSION IN LIVING CELLS

Citation
Rn. Day et al., DUAL-FUNCTION REPORTER PROTEIN FOR ANALYSIS OF GENE-EXPRESSION IN LIVING CELLS, BioTechniques, 25(5), 1998, pp. 848
Citations number
28
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Biochemical Research Methods
Journal title
BioTechniques → ACNP
ISSN journal
0736-6205
Volume
25
Issue
5
Year of publication
1998
Database
ISI
SICI code
0736-6205(1998)25:5<848:DRPFAO>2.0.ZU;2-H
Abstract
The firefly luciferase (Luc) protein and the jellyfish green fluoresce nt protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Lire useful for a particular experimental application are quite dis tinct. A recombinant protein with both fluorescent ann bioluminescent characteristics might take advantage of the strengths of both reporter s. An expression vector encoding a chimeric protein in which GFP was t ethered to Luc through a 19-amino acid linker was prepared and charact erized. Western blotting with antibodies specific for either GFP or Lu c showed that a protein of appropriate size was expressed in transfect ed cells. Fluorescence microscopy revealed bright gr een fluorescence from transfected cells, indicating proper formation of the GFP chromop hore. Lire enzymatic activity in protein extracts from transfected cel ls showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the h alf-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantific ation of Luc activity from the same living cells.