Cell Fusion pp 457-478 | Cite as

Microinjection of Culture Cells via Fusion with Loaded Erythrocytes

  • Robert A. Schlegel
  • Michael R. Lieber


Because of its relative simplicity, the non-nucleated mammalian erythrocyte has often served as a paradigm for study of more complex eukaryotic cells. About 10 years ago, studies of the erythrocyte membrane and investigations employing erythrocytes as models for understanding cell fusion converged, resulting in the technique referred to as erythrocyte-mediated microinjection. The seminal studies of Seeman (1967) indicated that mac-romolecules could enter and be trapped inside erythrocytes whose membrane integrity was reversibly interrupted by a hypotonic shock treatment. Loyter and colleagues (Peretz et al., 1974) then described conditions under which erythrocytes could be fused with one another by Sendai virus without losing their membrane integrity, so that intracellular mac-romolecules were retained. Under these same sorts of conditions hemoglobin could be transferred from erythrocytes to nucleated eukaryotic cells to which they were fused (Zakai et al., 1974; Furusawa et al., 1974). Finally, these findings on loading of erythrocytes with exogenous mac-romolecules and fusion of such carriers to recipient cells were combined to afford an efficient method for introducing macromolecules into eu-karyotic cells (Schlegel and Rechsteiner, 1975; Loyter et al., 1975; Nishimura et al., 1976).


Erythrocyte Membrane Fluorescence Recovery After Photobleaching Sendai Virus Erythrocyte Ghost Human Erythrocyte Membrane 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Antman, K. H., and Livingston, D. M., 1980, Intracellular neutralization of SV40 tumor antigens following microinjection of specific antibody, Cell 19:627–635.PubMedCrossRefGoogle Scholar
  2. Backer, J. M., Bourret, L., and Dice, J. F., 1983, Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids, Proc. Natl Acad. Sci. U.S.A. 80:2166–2170.PubMedCrossRefGoogle Scholar
  3. Baker, R. F., 1967, Ultrastructure of the red blood cell, Fed. Proc. 26:1785–1801.PubMedGoogle Scholar
  4. Bennett, F., Busch, H., Lischwe, M. A., and Yeoman, L. C., 1983, Antibodies to a nucleolar protein are localized in the nucleolus after red cell-mediated microinjection, J. Cell Biol. 97:1556–1572.CrossRefGoogle Scholar
  5. Bjerrum, P. J., 1979, Hemoglobin-depleted human erythrocyte ghosts: Characterization of morphology and transport functions, J. Membrane Biol. 48:43–46.CrossRefGoogle Scholar
  6. Bodeman, H., and Passow, H., 1972, Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis, J. Membrane Biol. 8:1–26.CrossRefGoogle Scholar
  7. Boogaard, C., and Dixon, G. H., 1983a, Red cell ghost-mediated microinjection of RNA into HeLa cells. I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA, Exp. Cell Res. 143:175–190.PubMedCrossRefGoogle Scholar
  8. Boogaard, C., and Dixon, G. H., 1983b, Red cell ghost-mediated microinjection of RNA into HeLa cells: II. Cellular translation of protamine mRNA; post-translational modifications and nuclear binding of newly-synthesized protamine, Exp. Cell Res. 43:191–205.CrossRefGoogle Scholar
  9. Brown, D. B., Hanks, S. K., Murphy, E. C., and Rao, P. N., 1985, Early initiation of DNA synthesis in Gl phase HeLa cells following fusion with red cell ghosts loaded with S-phase extracts, Exp. Cell Res. 156:251–259.PubMedCrossRefGoogle Scholar
  10. Capecchi, M. R., Von der Haar, R. A., Capecchi, N. E., and Sveda, M. M., 1977, The isolation of a suppressable nonsense mutant in mammalian cells, Cell 12:371–381.PubMedCrossRefGoogle Scholar
  11. Celis, J. E., 1984, Microinjection of somatic cells with micropipets: Comparison with other transfer techniques, Biochem. J. 223:281–291.PubMedGoogle Scholar
  12. Celis, J. E., Kaltoft, K., and Bravo, R., 1980, Microinjection of somatic cells, in: Introduction of Macromolecules into Viable Mammalian Cells (R. Baserga, C. Croce, and G. Rovera, eds.), pp. 99–123, Alan Liss, New York.Google Scholar
  13. Danon, D., 1961, Osmotic hemolysis by gradual decrease in the ionic strength of the surrounding medium, J. Cell Comp. Physiol. 57:111–117.PubMedCrossRefGoogle Scholar
  14. Davies, H. G., Marsden, N. V. B., Ostling, S. G., and Zade-Oppen, A. M. M., 1968, The effect of some neutral macromolecules on the pattern of hypotonic hemolysis, Acta Physio. Scand. 74:577–593.CrossRefGoogle Scholar
  15. Dice, J. F., 1982, Altered degradation of proteins microinjected into senescent human fibroblasts, J. Biol. Chem. 257:14624–14627.PubMedGoogle Scholar
  16. Fowler, V., and Branton, D., 1981, Lateral mobility of human erythrocyte integral membrane proteins, Nature (Lond.) 268:23–26.CrossRefGoogle Scholar
  17. Frye, L. D., and Edidin, M., 1970, Rapid mixing of cell surface antigens after formation of mouse-human heterokaryons, J. Cell Sci. 7:319–335.PubMedGoogle Scholar
  18. Furusawa, M., 1980, Cellular microinjection by cell fusion: Technique and applications in biology and medicine, Int. Rev. Cytol. 62:29–67.PubMedCrossRefGoogle Scholar
  19. Furusawa, M., Nishimura, T., Yamaizumi, M., and Okada, Y., 1974, Injection of foreign substances into animal cells by fusion, Nature (Lond.) 249:449–450.CrossRefGoogle Scholar
  20. Furusawa, M., Yamaizumi, M., Nishimura, T., Uchida, T., and Okada, Y., 1976, Use of erythrocyte ghosts for injection of substances into animal cells by cell fusion, Methods Cell Biol. 14:73–80.PubMedCrossRefGoogle Scholar
  21. Godfrey, W., Doe, B., and Wofsy, L., 1983, Immunospecific vesicle targeting facilitates microinjection into lymphocytes, Proc. Natl. Acad. Sci. U.S.A. 80:2267–2271.PubMedCrossRefGoogle Scholar
  22. Heedman, P. A., 1958, Hemolysis of individual red blood cells, Exp. Cell Res. 14:9–22.PubMedCrossRefGoogle Scholar
  23. Hendil, K. B., 1980, Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells, J. Cell. Physiol. 105:449–460.PubMedCrossRefGoogle Scholar
  24. Heumann, R., Schwab, M., and Theonen, H., 1981, A second messenger required for nerve growth factor biological activity?, Nature (Lond.) 292:838–840.CrossRefGoogle Scholar
  25. Honda, K., Maeda, Y., Sasakawa, J., Ohno, H., and Tsuchida, E., 1981, Activities of cell fusion and lysis of the hybrid type of chemical fusogens. I. Structure and function of the promoter of cell fusion, Biochem. Biophys. Res. Commun. 100:442–448.PubMedCrossRefGoogle Scholar
  26. Huhn, D., Pauli, G. D., and Grassmann, D., 1970, Die Erythrocytenmembran: Feinstruktur der gefrierfeatzten Membran nach Einwirkung von hyptonen Lösungen und Saponin, Klin. Wochenschr. 48:939–943.PubMedCrossRefGoogle Scholar
  27. Hui, S. W., Isac, T., Boni, L. T., and Sen, A., 1985, Action of polyethylene glycol on the fusion of human erythrocyte membranes, J. Membrane Biol. 84:137–146.CrossRefGoogle Scholar
  28. Iino, T., Furusawa, I., and Obinata, M., 1983, Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection, Exp. Cell Res. 148:475–480.PubMedCrossRefGoogle Scholar
  29. Johnson, R. M., Taylor, G., and Meyer, D. B., 1980, Shape and volume changes in erythrocyte ghosts and spectrin-actin networks, J. Cell Biol. 86:371–376.PubMedCrossRefGoogle Scholar
  30. Jonak, G. J., and Mora, M., 1980, Rapid identification of microinjected mammalian cells by fusion with loaded chicken erythrocytes, in: Introduction of Macromolecules into Viable Mammalian Cells (R. Baserga, C. Croce, and G. Rovera, eds.), pp. 157–167, Alan Liss, New York.Google Scholar
  31. Kaltoft, K., and Celis, J. E., 1978, Ghost mediated transfer of human hypoxanthineguanine phosphoribosyl transferase into deficient Chinese hamster ovary cells by means of polyethylene glycol-induced fusion, Exp. Cell Res. 115:423–428.PubMedCrossRefGoogle Scholar
  32. Kaltoft, K., Zeuthen, J., Engberg, F., Piper, P. W., and Celis, J. E., 1976, Transfer of tRNAs to somatic cells mediated by Sendai virus-induced fusion, Proc. Natl Acad. Sci. U.S.A. 73:2793–2797.PubMedCrossRefGoogle Scholar
  33. Kriegler, M. P., and Livingston, D. M., 1977, Chemically facilitated microinjection of protein into intact monolayers of tissue culture cells, Somat. Cell Genet. 3:603–610.PubMedCrossRefGoogle Scholar
  34. Kriegler, M. P., Griffen, J. D., and Livingston, D. M., 1978, Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen, Cell 14:983–994.PubMedCrossRefGoogle Scholar
  35. Kruse, C. A., Spector, E. B., Cederbaum, S. D., Wisnieski, B. J., and Popjack, G., 1981, Microinjection of arginase into enzyme deficient cells with the isolated glycoproteins of Sendai virus as fusogen, Biochem. Biophys. Acta 645:339–345.PubMedCrossRefGoogle Scholar
  36. Kruse, C. A., Wisnieski, B. J., and Popjack, G., 1984, Characterization of a glycoprotein fusogen isolated from Sendai virus, Biochem. Biophys. Acta 797:40–50.PubMedCrossRefGoogle Scholar
  37. Kulka, R. G., and Loyter, A., 1979, The use of fusion methods for the microinjection of animal cells, Curr. Topics Membrane Trans. 12:365–430.CrossRefGoogle Scholar
  38. Lange, Y., Hadesman, R. A., Steck, T. L., 1982a, Role of the reticulum in stability of the isolated human erythrocyte membrane, J. Cell Biol. 92:714–721.PubMedCrossRefGoogle Scholar
  39. Lange, Y., Gough, A., and Steck, T. L., 1982b, Role of the bilayer in the shape of the isolated erythrocyte membrane, J. Membrane Biol. 69:113–123.CrossRefGoogle Scholar
  40. Lieber, M. R., 1981, Characterization of the hemolytic holes in human erythrocyte membranes, Ph.D. dissertation, University of Chicago.Google Scholar
  41. Lieber, M. R., and Steck, T. L., 1982a, A description of the holes in human erythrocyte membrane ghosts, J. Biol. Chem. 257:11651–11659.PubMedGoogle Scholar
  42. Lieber, M. R., and Steck, T. L., 1982b, Dynamics of the holes in human erythrocyte membrane ghosts, J. Biol. Chem. 257:11660–11666.PubMedGoogle Scholar
  43. Loyter, A., Zakai, N., and Kulka, R. G., 1975, “Ultramicroinjection” of macromolecules or small particles into animal cells, J. Cell Biol. 66:292–304.PubMedCrossRefGoogle Scholar
  44. Maeda, T., and Ohnishi, S., 1980, Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes, FEBS Lett. 122:283–287.PubMedCrossRefGoogle Scholar
  45. McElligott, M. A., and Dice, J. F., 1983, Erythrocyte-mediated microinjection: A technique to study protein degradation in muscle cells, Biochem. J. 216:559–566.PubMedGoogle Scholar
  46. McElligott, M. A., and Dice, J. F., 1984, Microinjection of cultured cells using red-cell-mediated fusion and osmotic lysis of pinosomes: A review of methods and applications, Biosci. Rep. 4:451–466.PubMedCrossRefGoogle Scholar
  47. Mekada, E., Yamaizumi, M., and Okada, Y., 1978a, An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II), J. Histochem. Cytochem. 26:62–67.PubMedCrossRefGoogle Scholar
  48. Mekada, E., Yamaizumi, M., Uchida, T., and Okada, Y., 1978, Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter, J. Histochem. Cytochem. 26:1067–1073.PubMedCrossRefGoogle Scholar
  49. Mercer, W. E., 1980, Intracellular factor(s) involved in the control of mammalian cell proliferation, Ph.D. dissertation, Pennsylvania State University.Google Scholar
  50. Mercer, W. E., and Baserga, R., 1982, Techniques for decreasing the toxicity of polyethylene glycol, in: Techniques in Somatic Cell Genetics (J. W. Shay, ed.), pp. 23–34, Plenum Press, New York.CrossRefGoogle Scholar
  51. Mercer, W. E., Terefinko, D. J., and Schlegel, R. A., 1979, Red cell-mediated microinjection of macromolecules into monolayer cultures of mammalian cells, Cell Biol Int. Rep. 3:265–271.PubMedCrossRefGoogle Scholar
  52. Neff, N. J., Bourret, L., Maio, P., and Dice, J. F., 1981, Degradation of proteins microinjected into IMR-90 human diploid fibroblasts, J. Cell Biol. 91:184–194.PubMedCrossRefGoogle Scholar
  53. Nishimura, T., Furusawa, M., Yamaizumi, M., and Okada, Y., 1976, Method for intracellular injection by cell fusion using erythrocyte ghosts, Cell Struct. Funct. 1:197–200.CrossRefGoogle Scholar
  54. Ohara, J., and Watanabe, T., 1982, Microinjection of macromolecules into normal murine lymphocytes by cell fusion technique, J. Immunol. 128:1090–1096.PubMedGoogle Scholar
  55. Peretz, H., Toister, Z., Laster, Y., and Loyter, A., 1974, Fusion of intact human erythrocytes and erythrocyte ghosts, J. Cell Biol. 63:1–11.PubMedCrossRefGoogle Scholar
  56. Petrie, H. T., and McDonel, J. L., and Schlegel, R. A., 1982, Intracellular antibody to Clostridium perfringens enterotoxin fails to counteract toxin-induced damage, Cell Biol. Int. Rep. 6:705–711.PubMedCrossRefGoogle Scholar
  57. Rechsteiner, M., 1975, Uptake of proteins by red blood cells, Exp. Cell Res. 93:487–492.PubMedCrossRefGoogle Scholar
  58. Rechsteiner, M. C., 1982, Transfer of macromolecules using erythrocyte ghosts, in: Techniques in Somatic Cell Genetics (J. W. Shay, ed.), pp. 385–398, Plenum Press, New York.CrossRefGoogle Scholar
  59. Rechsteiner, M., and Kuehl, L., 1979, Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells, Cell 16:901–908.PubMedCrossRefGoogle Scholar
  60. Rechsteiner, M. C., and Schlegel, R. A., 1986, Erythrocyte-mediated transfer: Applications, in: Microinjection and Organelle Transplantation Techniques: Methods and Applications (J. E. Celis, A. Graessman, and A Loyter, eds.), pp. 89–116, Academic Press, New York.Google Scholar
  61. Rechsteiner, M., Wu, L. H., and Miller, A. O. A., 1984, RBC-mediated microinjection of chromatin components into cultured mammalian cells, in: Red Blood Cells as Carriers for Drugs (J. R. DeLoach and U. Sprandel, eds.), pp. 142–149, Karger, Basel.Google Scholar
  62. Sambrook, J., Rogers, L., White, J., and Gething, M. J., 1985, Lines of murine cells that constituitively express influenza virus hemagglutinin, EMBO J. 4:91–103.PubMedGoogle Scholar
  63. Schlegel, R. A., 1984, Red cell-mediated microinjection of antibodies, in: Red Blood Cells as Carriers for Drugs (J. R. DeLoach and U. Sprandel, eds.), pp. 134–141, Karger, Basel.Google Scholar
  64. Schlegel, R. A., and McEvoy, L., 1986, Red cell-mediated microinjection of proteins and nucleic acids, Methods Enzymol. (in press).Google Scholar
  65. Schlegel, R. A., and Mercer, W. E., 1980, Red cell-mediated microinjection of quiescent fibroblasts, in: Introduction of Macromolecules into Viable Mammalian Cells (R. Baserga, C. Croce, and G. Rovera, eds.), pp. 145–155, Alan Liss, New York.Google Scholar
  66. Schlegel, R. A., and Rechsteiner M. C., 1975, Microinjection of thymidine kinase and bovine serum albumin into mammalian cells by fusion with red blood cells, Cell 5:371–379.PubMedCrossRefGoogle Scholar
  67. Schlegel, R. A., and Rechsteiner, M. C., 1978, Red cell-mediated microinjection of macromolecules into mammalian cells. Methods Cell Biol. 20:341–354.PubMedCrossRefGoogle Scholar
  68. Schlegel, R. A., and Rechsteiner, M. C., 1986, Erythrocyte-mediated transfer: Methods, in: Microinjection and Organelle Transplantation Techniques: Methods and Applications (J. E. Celis, A Graessman, and A. Loyter, eds.), pp. 67–87, Academic Press, New York.Google Scholar
  69. Schlegel, R. A., and Lumley-Sapanski, K., and Williamson, P., 1985a, Insertion of lipid domains into plasma membranes by fusion with erythrocytes, Biochem. Biophys. Acta 846:234–241.PubMedCrossRefGoogle Scholar
  70. Schlegel, R. A., Miller, L. S., and Rose, K. M., 1985b, Reduction in RNA synthesis following red cell-mediated microinjection of antibodies to RNA polymerase I, Cell Biol. Intl. Rep. 9:341–350.CrossRefGoogle Scholar
  71. Schneiderman, S., Farber, J. L., and Baserga, R., 1979, Techniques for decreasing the toxicity of polyethylene glycol, Somat. Cell Genet. 5:263–269.PubMedCrossRefGoogle Scholar
  72. Seeman, P., 1967, Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin, J. Cell Biol 32:55–70.PubMedCrossRefGoogle Scholar
  73. Seeman, P., 1973, Macromolecules may inhibit diffusion of hemoglobin from lysing erythrocytes by exclusion of solvent, Can. J. Physiol Pharmacol. 51:226–229.PubMedCrossRefGoogle Scholar
  74. Seeman, P., 1974, Ultrastructure of membrane lesions in immune lysis, osmotic lysis, and drug-induced lysis, Fed. Proc. 33(10):2116–2134.PubMedGoogle Scholar
  75. Seeman, P., Cheng, D., and Iles, G. H., 1973, Structure of membrane holes in osmotic and saponin hemolysis, J. Cell Biol. 56:519–527.PubMedCrossRefGoogle Scholar
  76. Smith, C. L., Ahkong, Q. F., Fisher, D., Lucy, J. A., 1982, Is purified polyethylene glycol able to induce cell fusion?, Biochem. Biophys. Acta 692:109–114.PubMedCrossRefGoogle Scholar
  77. Straus, S. E., and Raskas, H. J., 1980, Transfection of KB cells by polyethylene glycol-induced fusion with erythrocyte ghosts containing adenovirus type 2 DNA, J. Gen. Virol. 48:241–245.PubMedCrossRefGoogle Scholar
  78. Wasserman, M., Zakai, N., Loyter, A., and Kulka, R. G., 1976, A quantitative study of ul-tramicroinjection of macromolecules into animal cells, Cell 7:551–556.PubMedCrossRefGoogle Scholar
  79. White, J., Helenius, A., and Kartenbeck, J., 1982, Membrane fusion activity of influenza, EMBO J. 1:217–222.PubMedGoogle Scholar
  80. Wiberg, F. C., Sunnerhagen, P., Kaltoft, K., Zeuthen, J., and Bjursell, G., 1983, Replication and expression in mammalian cells of transfected DNA: Description of an improved erythrocyte ghost fusion technique, Nucl. Acids Res. 11:7287–7302.PubMedCrossRefGoogle Scholar
  81. Wojcieszyn, J. W., Schlegel, R. A., Lumley-Sapanski, K., and Jacobson, K A., 1983, Studies of the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes, J. Cell Biol. 96:151–159.PubMedCrossRefGoogle Scholar
  82. Wu, L., Kuehl, L., and Rechsteiner, M., 1981, Comparative studies on microinjected high mobility group chromosomal proteins, HMG1 and HMG2, J. Cell Biol 91:488–496.PubMedCrossRefGoogle Scholar
  83. Yamaizumi, M., Furusawa, M., Uchida, T., Nishimura, T., and Okada, Y., 1978a, Characterization of the ghost fusion method: A method for introducing exogenous substances into cultured cells, Cell Struct. Fund. 3:293–304.CrossRefGoogle Scholar
  84. Yamaizumi, M., Uchida, T., Okada, Y., and Furusawa, M., 1978b, Neutralization of Diptheria toxin in living cells by microinjection of antifragment A contained within resealed erythrocyte ghosts, Cell 13:227–232.PubMedCrossRefGoogle Scholar
  85. Yamaizumi, M., Mekada, E., Uchida, T., and Okada, Y., 1978c, One molecule of Diptheria toxin fragment A introduced into a cell can kill the cell, Cell 15:245–250.PubMedCrossRefGoogle Scholar
  86. Yamaizumi, M., Uchida, T., Okada, Y., Furusawa, M., and Mitsui, H., 1978d, Rapid transfer of non-histone chromosomal proteins to the nucleus of living cells, Nature (Lond.) 273:783–784.CrossRefGoogle Scholar
  87. Yamaizumi, M., Uchida, T., Mekada, E., and Okada, Y., 1979, Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells, Cell 18:1009–1014.PubMedCrossRefGoogle Scholar
  88. Yee, J. P., and Mel, H. C., 1978, Cell-membrane and rheological mechanisms: Dynamic osmotic hemolysis of human erythrocytes and repair of ghosts, as studied by resistive pulse spectroscopy, Biorheology 15:321–339.PubMedGoogle Scholar
  89. Zade-Oppen, A M. M., Ostling, S. G., and Marsden, N. V. B., 1979, On how macromolecules reduce hemoglobin loss in hypotonic hemolysis, Upsula J. Med. Sci. 84:155–161.CrossRefGoogle Scholar
  90. Zakai, N., Loyter, A., and Kulka, R., 1974, Fusion of erythrocytes and other cells with retention of erythrocyte cytoplasm: Nuclear activation in chicken erythrocyte-melanoma heterokaryons, J. Cell Biol. 61:241–248.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1987

Authors and Affiliations

  • Robert A. Schlegel
    • 1
  • Michael R. Lieber
    • 2
  1. 1.Department of Molecular and Cell BiologyPennsylvania State UniversityUSA
  2. 2.Laboratory of PathologyNational Institutes of HealthBethesdaUSA

Personalised recommendations