ABSTRACT
The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated alpha-Poxn2 and alpha-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The alpha-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.
Subject(s)
Drosophila Proteins , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Nerve Tissue Proteins/immunology , Transcription Factors , Animals , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Cloning, Molecular , Drosophila/chemistry , Escherichia coli/genetics , Evaluation Studies as Topic , Mice , Mice, Inbred NZB , Mice, Inbred Strains , Nerve Tissue Proteins/genetics , Paired Box Transcription Factors , Recombinant Proteins/isolation & purificationABSTRACT
Intrabodies show great promise for controlling gene expression. As an initial attempt to evaluate the intrabody technology in Drosophila, the gene poxn was used as target. Transgenic flies harboring different anti-Poxn scFv genes integrated into various chromosomes were obtained. In one transformant, a phenocopy resembling the hypomorphic poxn-phenotype was produced in embryos and larvae following induction of expression of alpha-Poxn2 intrabody. The antisense approach was used as control. Parameters that can affect the success of intrabody technology are described.
