Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 3655-3659, May 1990 Biochemistry Receptor-mediated endocytosis of transferrin-polycation conjugates: An efficient way to introduce DNA into hematopoietic cells (transferrinfection/transferrin receptor/chloroquine/DNA transfection/gene therapy) MARTIN ZENKE, PETER STEINLEIN, ERNST WAGNER, MATTHEW COTTEN, HARTMUT BEUG, AND MAX L. BIRNSTIEL Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria Contributed by Max L. Birnstiel, February 15, 1990 ABSTRACT Most current gene transfer methods function via the transferrin cycle (a method termed "transferrinfec- satisfactorily in specialized systems involving established cell tion"). Pilot experiments described in the previous commu- lines but are often not applicable with nonadherent, primary nication, showing the principal feasibility of the transferrin- hematopoietic cells, which are notoriously difficult to transfect. polylysine gene transfer approach (8), encouraged us to study To approach this problem, we have investigated an alternative its usefulness with hematopoietic cells in more detail, by method of gene transfer, "transferrnfection," in which DNA using both established cell lines and primary hematopoietic complexed to transferrin-polycation conjugates is introduced cells of avian origin. into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that MATERIALS AND METHODS transferrin-polylysine and transferrin-protamine, when com- Transferrin-Polylysine and Transferrin-Protamine Conju- plexed to plasmid DNA containing a luciferase reporter gene, gates and Recombinant Plasmids. The origin of polylysine is efficiently bound and moved into avian erythroblasts by (Sigma), protamine (Sigma), and chicken transferrin (ion- endocytosis. Successful transfer and expression of the Iu- saturated conalbumin; Sigma) and the synthesis of transfer- ciferase reporter gene depends on specific interaction of the rin-polylysine and transferrin-protamine conjugates have transferrin-polylysine-DNA complex with the transferrin re- been described (8). The pRSVL plasmid DNA (10) as well as ceptor and occurs in a significant fraction (>95%) of the cells. the pRSV-,8Gal and pB-SK- DNAs (Stratagene) were pre- Gene transfer efficiency by transferrinfection is lower than pared by using the Triton-X lysis procedure (11) followed by with an optimized DEAE-dextran transfection method but cesium chloride/ethidium bromide equilibrium density gra- reaches similar efficiencies when the cells are treated with dient centrifugation, destaining with 1-butanol, and dialysis chloroquine. Because the procedure in the absence of chloro- against 10 mM Tris HCl, pH 7.5/1 mM EDTA. quine is completely nontoxic to cells, a constant expression level Cells and Cell Culture. The temperature-sensitive v- of transferred genes may be maintained by repeated additions erbB-transformed chicken erythroblast cell line HD3 (12) as of transferrin-polylysine-DNA complex. In addition, the use- well as the lymphoid (REVT) transformed clone REV NPB4 fulness of transferrinfection for gene transfer into primary (13) were grown in standard growth medium (EBM + H; ref. hematopoietic cells is demonstrated. 12) at a density of 1-2 x 106 cells per ml at 37°C and 5% Co2. A primary clone of erythroblasts transformed by a retrovirus Many techniques are available to introduce and express expressing the human epidermal growth factor receptor to- foreign DNA in eukaryotic cells (1-4). However, most pro- gether with a temperature-sensitive myb oncogene (EGFR- cedures function well mainly with adherent cells and/or ts-myb) was generated as will be described elsewhere (ref. 14 established cell lines and are often associated with severe and H.B., unpublished work). The cells were grown in cytotoxic side effects. In particular, most available methods CFU-E medium (15) in the presence of 20 ng of epidermal proved to be ineffective or too toxic with nonestablished growth factor per ml. Normal bone marrow cells were hematopoietic cells. Only recently, specific protocols em- prepared as described (16) and enriched for immature ery- ploying electroporation or DEAE-dextran have met with throid cells by centrifugation through Percoll at 1.072 g/cm3 some success in such cells (5, 6). We have therefore studied and removal of cells adherent to plastic (17). the utility of receptor-mediated endocytosis as an alternative Transferrin-Polylysine-DNA or Transferrin-Protamine- means to introduce and express DNA in hematopoietic cells, DNA Complex Formation and Gene Transfer Reaction. In a thus avoiding cytotoxic effects. A similar approach has been typical complex formation reaction 10 ,g of transferrin- used by Wu and Wu (7) who employed the asialoglycoprotein polylysine or transferrin-protamine conjugate in 250 ,ul of receptor to deliver DNA to liver cells. H20 was added to 3 ,tg of pRSVL plasmid DNA contained In a recent communication (8) we have described trans- in 250 ,lI of 0.3 M NaCl (added while agitating). Phosphate ferrin-polycation conjugates that efficiently bind DNA of buffers should be avoided because precipitates form. By widely different size and can replace native iron transferrin as using the above conditions, up to 100 ,ug of transferrin- an efficient iron transporter into the living cell. Because polylysine or transferrin-protamine conjugate and 30 gg of transferrin receptors are elevated on the surface of exten- plasmid DNA could be used per 500 ,ul of final volume sively proliferating or neoplastic hematopoietic cells (for without precipitation of the transferrin-polylysine-DNA or review, see ref. 9), we attempted to use these transferrin- transferrin-protamine-DNA complexes. After 30 min at polycation conjugates to efficiently introduce DNA into cells room temperature, the transferrin-polylysine-DNA or trans- ferrin-protamine-DNA mixture was directly added to 5-10 x The publication costs of this article were defrayed in part by page charge 106 HD3 cells growing at 0.5-1 x 106 cells per ml in standard payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: FITC, fluorescein isothiocyanate. 3655 Downloaded by guest on September 29, 2021 3656 Biochemistry: Zenke et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1.c4 growth medium (see above) and incubated for 16-48 hr. B Other cell types were treated similarly, except being grown 1.c.2 -A / in their respective media [see above; CFU-E medium plus 1, REV factor (13) was used for the normal bone marrow cells]. .8 P. 0) Preparation of Tritiated Transferrin-Polylysine. Tritiated .6- F, transferrin-polylysine was prepared by conjugation of triti- ated polylysine to transferrin in a similar fashion as described m0.0..4- E (8). Tritiation of poly(L-lysine) (hydrobromide salt, Mr .2 z 18,000; Sigma) was performed by treatment with formalde- 0 hyde and tritiated sodium borohydride (18). 0 20 40 60 80 100 120 1 10 100 1000 Uptake of Transferrin-Polylysine-DNA Complexes into In- Total relative fluorescence tracellular Vesicles. Avian erythroblasts (HD3; ref. 12) were intensity incubated with transferrin-free differentiation medium (8) at FIG. 1. Binding and internalization of transfemn-polylysine or 370C for 18 hr. After addition of transferrin-polylysine conju- Tritiated transferrin- at the transferrin-polylysine-DNA by HD3 cells. (A) gates [labeled with fluorescein isothiocyanate (FITC) polylysinego (r) or tritiated transferrin-polylysinego-DNA (A) were polylysine moiety (8)] that had been complexed with DNA in assayed for specific binding to the transferrin receptor of HD3 cells, some of the experiments, cells were further incubated for 18 as described. (B) HD3 cells were incubated with FITC-transferrin at hr. Cells were cytocentrifuged onto slides, fixed with a mixture 40 ,ug/ml (-e-), FITC-transferrin-polylysine270 at 50 ,Lg/ml (-), of 3.7% formaldehyde (prepared from paraformaldehyde; ref. FITC-transferrin-polylysine270 plus pB-SK- DNA at 50 ,ug/ml and 19)/0.02% glutaraldehyde, washed with phosphate-buffered 16 jig/ml, respectively (->e), and binding buffer (.) for 6 hr and processed for fluorescence-activated cell sorter analysis as de- saline (PBS), mounted in Mowiol 4.88 (Hoechst) (19), and scribed. examined using a Zeiss Axiophot fluorescence microscope. FITC-labeled goat antimouse antibody (0.1 mg/ml) was used observations were confirmed by FACSCAN analysis ofHD3 in the controls. For quantitative determination of FITC- cells allowed to internalize FITC-transferrin-polylysine or transferrin or FITC-transferrin-polylysine-DNA, cells were transferrin-polylysine-DNA for 6 hr. With both transferrin- incubated with the respective transferrin-polylysine prepara- polylysine and transferrin-polylysine-DNA, essentially all tion for 6 hr at 370C, washed three times in cold PBS/bovine in- serum albumin, and processed for quantitative fluorescence cells showed >10-fold increase in relative fluorescence activated cell sorter analysis in a Becton Dickinson FACScan. tensity (Fig. 1B), indicating that the transferrin-polylysine- Luciferase Assay. Aliquots from cultures receiving trans- DNA complexes are internalized by >95% of the cells. ferrin-polylysine/transferrin-protamine-DNA complexes Optimized Procedure for Transferrinfection of Avian Eryth- were harvested by centrifugation (8 min, 1500 X g, 4°C), cell roblasts. To establish an optimal ratio