Expression and targeting of intracellular antibodies in mammalian cells.
Genes encoding the heavy and light chains of a hapten-specific IgM antibody were modified by site-directed mutagenesis to destroy the hydrophobic leader sequences and allow expression in the cytoplasm of non-lymphoid cells. The in situ assembly of the mutant heavy and light chains was tested in transfected cell lines by immunofluorescence using anti-idiotypic antibodies. A positive diffuse cytoplasmic staining was observed. This demonstrated that the antibody polypeptide chains could assemble in the cell cytoplasm and led us to ask whether antibodies could be further targeted to the nucleus. Mutations were therefore made in which the leader sequence of the light chain was replaced by the nuclear localization signal of the SV40 large T antigen. Transfectants in which the heavy chain lacking the hydrophobic leader was expressed together with a light chain carrying the nuclear localization signal were selected and a nuclear distribution of the assembled antibody was found. Thus, it should prove possible to target a specific antibody to the cell nucleus with the aim of interfering with the function of a nuclear antigen.
Biocca
S
S
Institute of Neurobiology, CNR, Roma, Italy.
Neuberger
M S
MS
Cattaneo
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D002467:Cell Nucleus / Q000276:immunology; D003593:Cytoplasm / Q000276:immunology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006241:Haptens; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D013539:Simian virus 40 / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
D000957:Antigens, Viral, Tumor; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D021382:Protein Sorting Signals