Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells

Abstract

Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions.

Keywords: electroporation; intracellular delivery; intracellular imaging; living cells; monoclonal antibodies; protein interference.

Figure 1. Intracellular localization of delivered antibodies as probed by immunofluorescence microscopy. (A) Micrographs show typical HeLa cells transduced with either 4C6 or 7C2 antibody. The delivered antibodies were revealed with Alexa 448-labeled goat anti-mouse immunoglobulins 24 h post-transduction. Both antibodies are IgG1κ. Magnification: 630 × . (B) The pictures correspond to typical fields of HeLa cells transduced with the indicated antibodies at 24, 48 and 72 h post-treatment. The delivered antibodies were revealed as in A. The panels on the right correspond to the staining by indirect immunofluorescence of HeLa cells incubated with the relevant antibody (in vitro staining). Magnification: 630 × . (C) The pictures show typical nuclei of HeLa cells either transduced and fixed at 48 h post-treatment (in cells) or stained by indirect immunofluorescence (in vitro) with 2H3 antibody without (- UV) or with (+ UV) UVC irradiation 8 h before analysis. Magnification: 100 × .

Figure 2. Biological effect of delivered anti-E6 and anti-DNA polymerase α antibodies. (A) Micrographs of typical CaSki cells following transfection with anti-E6 siRNA or transduction with 4C6 antibody. At 48 h post-treatment, the 4C6 antibodies were revealed with Alexa 568-labeled goat anti-mouse immunoglobulins, in parallel with p53 as indicated in the method section. Magnification: 630 × . (B) Micrographs of HeLa cells either stained by indirect immunofluorescence (in vitro) or after transduction at 48 h (in cells) with the indicated antibody. The monoclonal antibodies were detected as indicated in the legend of Figure 1. Magnification: 630 × . (C) The panels on the left represent typical HeLa cells after transduction of 4E9-NLS and SJK123-NLS conjugates and stained with Alexa 488-labeled goat anti-mouse immunoglobulins as in B (in cells). Magnification: 630 × . The micrographs on the right represent typical fields of cells after 2 or 5 d post-treatment with each conjugate observed under the optical microscope. Typical enlarged nuclei are ticked with black arrows. Magnification: 200 × .