期刊
EUROPEAN JOURNAL OF CELL BIOLOGY
卷 79, 期 2, 页码 144-149出版社
URBAN & FISCHER VERLAG
DOI: 10.1078/S0171-9335(04)70016-3
关键词
GFP; expression; Paramecium; transformation
类别
资金
- NIGMS NIH HHS [GM 22714] Funding Source: Medline
In this paper we describe the expression of green fluoresent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV, Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.
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