Expression of the green fluorescent protein in Paramecium tetraurelia

Citation
K. Hauser et al., Expression of the green fluorescent protein in Paramecium tetraurelia, EUR J CELL, 79(2), 2000, pp. 144-149
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell & Developmental Biology
Journal title
EUROPEAN JOURNAL OF CELL BIOLOGY
ISSN journal
0171-9335 → ACNP
Volume
79
Issue
2
Year of publication
2000
Pages
144 - 149
Database
ISI
SICI code
0171-9335(200002)79:2<144:EOTGFP>2.0.ZU;2-V
Abstract
In this paper we describe the expression of green fluoresent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This i s not trivial because of the limited number of strong promoters available f or heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into accou nt differences in codon usage we selected and modified the original GFP ope n reading frame (ORF) from Aequorea victoria and placed the altered ORF int o the Paramecium expression vector pPXV, Injection of the linearized plasmi d into the macronucleus resulted in a cytoplasmic fluorescence signal in th e clonal descendants, which was proportional to the number of copies inject ed. Southern hybridization indicated the establishment and replication of t he plasmid during vegetative growth. Expression was also monitored by North ern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to s election with antibiotics and that it may also be used to construct and loc alize fusion proteins.