Targeting vectors for intracellular immunisation.
We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recombinant antibodies can be directed to different compartments of eukaryotic cells by means of previously defined targeting signals, thus permiting the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vectors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the localisation of scFvs to different intracellular compartments: the endoplasmic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondria (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins (scFvE-sec). The function of these vectors has been assessed by the immunofluorescence of COS cells transiently transfected with constructs containing the alphaD11 scFv.
Persic
L
L
Societa Italiana per la Ricerca Scientifica, Roma, Italy.
Righi
M
M
Roberts
A
A
Hoogenboom
H R
HR
Cattaneo
A
A
Bradbury
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002467:Cell Nucleus / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D008928:Mitochondria / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv