Mouse blastocyst immunosurgery with commercial antiserum to mouse erythrocytes

  • Y. P. Cruz
  • R. S. Treichel
  • E. Harsay
  • K. D. Chi
Growth, Differentiation, And Senescence


Immunosurgery is a useful technique for the isolation of inner cell masses from murine blastocysts. Conventionally, rabbit antisera made ad hoc against murine splenic or fetal cells or fibroblasts have been used as antibody sources. We investigated the feasibility of using commercially available rabbit antiserum to murine erythrocytes (anti-RBC) and compared it with rabbit antiserum generated ad hoc to murine L-cells (anti-L-cell). Our results indicate that anti-RBC is at least as effective as anti-L-cell serum for the immunosurgical isolation of inner cell masses, which became either miniblastocysts (later forming outgrowths) or embryoid bodies (undergoing ectoderm-endodermlike differentiation within 48 h). Because anti-RBC is commercially available, the technical modification described herein increases the accessibility of the immunosurgical protocol for the isolation of murine inner cell masses.

Key words

antibody cytotoxicity immunosurgery inner cell mass isolation 


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  1. Barends, P. M. G.; Stroband, H. W. J.; Taverne, N., et al. Integrity of the preimplantation pig blastocyst during expansion and loss of polar trophectoderm (Rauber cells) and the morphology of the embryoblast as an indicator for developmental stage. J. Reprod. Fertil. 87:715–726; 1989.PubMedCrossRefGoogle Scholar
  2. Barton, S. C.; Adams, C. A.; Norris, M. L., et al. Development of gynogenetic and parthenogenetic inner cell mass and trophectoderm tissues in reconstituted blastocysts in the mouse. J. Embryol. Exp. Morphol. 90:267–285; 1985.PubMedGoogle Scholar
  3. Chisholm, J. C.; Johnson, M. H.; Warren, P. D., et al. Developmental viability within and between mouse expanding blastocysts and their ICMs. J. Embryol. Exp. Morphol. 86:311–336; 1985.PubMedGoogle Scholar
  4. Copp, A. J. Interaction between inner cell mass and trophectoderm of the mouse blastocyst. I. A study of cellular proliferation. J. Embryol. Exp. Morphol. 48:109–125; 1978.PubMedGoogle Scholar
  5. Cruz, Y. P.; Pedersen, R. A. Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers. Dev. Biol. 112:73–83; 1985.PubMedCrossRefGoogle Scholar
  6. Eistetter, H. R. Pluripotent embryonal stem cell lines can be established from disaggregated mouse morulae. Dev. Growth Differ. 31:275–282; 1989.CrossRefGoogle Scholar
  7. Handyside, A. H. Time of commitment of inside cells isolated from preimplantation mouse embryos. J. Embryol. Exp. Morphol. 45:37–53; 1978.PubMedGoogle Scholar
  8. Handyside, A. H.; Hunter, S. Cell division and death in the mouse blastocyst before implantation. Roux’s Arch. Dev. Biol. 195:519–526; 1986.CrossRefGoogle Scholar
  9. Hogan, B.; Costantini, F.; Lacy, E. Manipulating the mouse embryo. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1986:106, 276.Google Scholar
  10. Hogan B.; Tilly, R.In vitro culture and differentiation of normal mouse blastocysts. Nature 265:626–629; 1977.PubMedCrossRefGoogle Scholar
  11. Hogan, B.; Tilly, R.In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. I. Inner cell masses from 3.5-dayp. c. blastocysts incubated for 24 h before immunosurgery. J. Embryol. Exp. Morphol. 45:93–105; 1978a.PubMedGoogle Scholar
  12. Hogan, B.; Tilly, R.In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts. II. Inner cell masses from 3.5- to 4.0-dayp. c. blastocysts. J. Embryol. Exp. Morphol. 45:107–121; 1978b.PubMedGoogle Scholar
  13. Klein, J. Immunology—the science of non-self discrimination. New York: John Wiley & Sons; 1982:65–68.Google Scholar
  14. Nichols, J.; Gardner, R. L. Heterogeneous differentiation of external cells in individual isolated early mouse inner cell masses in culture. J. Embryol. Exp. Morphol. 80:225–240; 1984.PubMedGoogle Scholar
  15. Norberg, H. S. Ultrastructural aspects of the preattached pig embryo: cleavage and early blastocyst stages. Z. Anat. EntwGesch. 143:94–114; 1973.CrossRefGoogle Scholar
  16. Papaioannou, V. E.; Ebert, K. M. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 102:793–803; 1988.PubMedGoogle Scholar
  17. Parr, E. L.; Moore, H. A. The fetal allograft: transplantation antigens on day 5 mouse blastocysts detected by an indirect but not by a direct immunoferritin procedure. In: Calaby, J. H.; Tyndale-Biscoe, C. H., eds. Reproduction and evolution. Canberra: Australian Academy of Sciences; 1977:363–364.Google Scholar
  18. Paul, W. E. Fundamental immunology, 2nd ed. New York: Raven Press; 1989:181–183.Google Scholar
  19. Solter, D.; Knowles, B. B. Immunosurgery of the mouse blastocyst. Proc. Natl. Acad. Sci. USA 72:5099–102; 1975.PubMedCrossRefGoogle Scholar
  20. Soltynska, M. S. Ultrastructure of mouse blastocysts during blastocoel expansion. Wilhelm Roux’s Arch. Dev. Biol. 194:425–428; 1985.CrossRefGoogle Scholar
  21. Spindle, A. Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos. J. Exp. Zool. 203:483–489; 1978.PubMedCrossRefGoogle Scholar
  22. Spindle, A. An improved culture medium for mouse blastocysts. In Vitro 16:669–674; 1980.PubMedCrossRefGoogle Scholar
  23. Spindle, A. Cell allocation in preimplantation mouse chimeras. J. Exp. Zool. 219:361–367; 1982.PubMedCrossRefGoogle Scholar
  24. Stites, D. P.; Stobo, J. D.; Wells, J. V., editors. Basic and clinical immunology, 6th ed. Norwalk, CT: Appleton and Lange; 1987:145–149.Google Scholar
  25. Wiley, L. M.; Spindle, A. I.; Pedersen, R. A. Morphology of isolated mouse inner cell masses developingin vitro. Dev. Biol. 63:1–10; 1978.PubMedCrossRefGoogle Scholar
  26. Winkel, G. K.; Pedersen, R. A. Fate of the inner cell mass in mouse embryos as studied by microinjection of lineage tracers. Dev. Biol. 127:143–156; 1988.PubMedCrossRefGoogle Scholar

Copyright information

© Tissue Culture Association 1993

Authors and Affiliations

  • Y. P. Cruz
    • 1
  • R. S. Treichel
    • 1
  • E. Harsay
    • 1
  • K. D. Chi
    • 1
  1. 1.Department of BiologyOberlin CollegeOberlin

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