Transgenic Research

, Volume 4, Issue 6, pp 353–360 | Cite as

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

  • Raymond L. Page
  • Stephen P. Butler
  • Anuradha Subramanian
  • Francis C. Gwazdauskas
  • John L. Johnson
  • William H. Velander


Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1∶1 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.


microinjection transgenesis polylysine DNA 


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  1. Anderson, G.B. (1992) Isolation and use of embryonic stem cells from livestock species.Animal Biotech. 3, 165–75.Google Scholar
  2. Behr, J.P., Demeneix, B., Loeffler, J.P. and Perez-Mutul, J. (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA.Proc. Natl Acad. Sci. USA 86, 6982–6.Google Scholar
  3. Biery, K.A., Bondioli, K.R. and De Mayo, F.J. (1988) Gene transfer by pronuclear injection in the bovine.Theriogenology 29, 224.Google Scholar
  4. Bloomfield, V.A. (1991) Condensation of DNA by multivalent cations: considerations of mechanism.Biopolymers 31, 1471–81.Google Scholar
  5. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid, DNA.Nucl. Acids Res. 7, 1513.Google Scholar
  6. Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. and Palmiter, R.D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjection.Proc. Natl Acad. Sci. USA 82, 4438–42.Google Scholar
  7. Campbell, S.M., Rosen, J.M., Hennighausen, L.G., Strech-Jurk, U. and Sippel, A.E. (1984) Comparison of the whey acidic protein genes of the rat and mouse.Nucl. Acids Res. 22, 8685–97.Google Scholar
  8. Canseco, R.S., Sparks, A.E.T., Pearson, R.E. and Gwazdauskas, F.C. (1992) Embryo density and medium volume effects on early embryo development.J. Assisted Reprod. Gen. 5, 454–7.Google Scholar
  9. Cotten, M., Langle-Rouault, F., Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H. and Bernsteil, M. (1990) Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.Proc. Natl Acad Sci. USA 87, 4033–7.Google Scholar
  10. Ebert, K.M., Selgrath, J.P., DiTullio, P., Denman, J., Smith, T.E., Memon, M.A., Schindler, J.E., Monastersky, G.M., Vitale, J.A. and Gordon, K. (1991) Transgenic production of a variant of human tissue-type plasminogen activator in goat milk: generation of transgenic goats and analysis of expression.Bio/Technology 9, 835–8.Google Scholar
  11. Evans, M.J. and Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos.Nature 292, 154–6.Google Scholar
  12. Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M. (1987) Lipofectin: a highly efficient, lipidmediated DNA-transfection procedure.Proc. Natl Acad. Sci. USA 84, 7413–7.Google Scholar
  13. Gerfen, R.W., Fajfar, C.J., and Wheeler, M.B. (1993) Isolation of embryonic cell lines from porcine blastocysts In: Bavister, B.D. ed.,Proc. Serono Symp. Preimplantation Development, p. 313. New York: Springer-Verlag.Google Scholar
  14. Hammer, R.E., Pursel, V.G., Rexroad, C.E. Jr., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D., and Brinster, R.L. (1985) Production of transgenic rabbits, sheep and pigs by microinjection.Nature 315, 680–3.Google Scholar
  15. Hogan, B., Costantini F., and Lacy, E. (1986)Manipulating the Mouse Embryo: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  16. Jaenisch, R. (1976) Germ line integration and mendelian transmission of the exogenous moloney leukemia virus.Proc. Natl Acad Sci. USA 73, 1260–4.Google Scholar
  17. Jaenisch, R. and Mintz, B. (1974) Simian virus 40 DNA sequences in healthy adult mice derived from preimplantation blastocysts injected with viral DNA.Proc. Natl Acad. Sci. USA 71, 1250–4.Google Scholar
  18. Laemmli, U.K. (1975) Characterization of DNA condensates induced by poly(ethylene oxide) and polylysine.Proc. Natl Acad. Sci. USA 72, 4288–92.Google Scholar
  19. Leng, M. and Felsenfeld, G. (1966) The preferential interactions of polylysine and polyarginine with specific base sequences in DNA.Biochem. J. 56 1325–32.Google Scholar
  20. Li, H.J., Chang, C. and Weiskopf, M. (1973) Helix-coil transition in nucleoprotein-chromatin structure.Biochem. J. 12, 1763–71.Google Scholar
  21. Loskutoff, N.M., Roessner, C.A. and Kraemer, D.C. (1986) Preliminary studies on liposome-mediated gene transfer: effects on survivability of murine zygotes.Theriogenology 25, 169.Google Scholar
  22. Reed, M.L., Roessner, C.A., Womack, J.E., Dorn, C.C. and Kraemer, D.C. (1988) Microinjection of liposome-encapsulated DNA into murine and bovine blastocysts.Theriogenology 29, 293.Google Scholar
  23. Saiki, R.K., Gelfand, D.J., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymeraseScience 239, 487–91.Google Scholar
  24. Salter, D.W., Smith, E.J., Hughes, S.H., Wright, S.E., Fadly, A.M., Witter, R.L. and Crittenden, L.B. (1986) Gene insertion into the chicken germ line by retroviruses.Poultry Sci. 65, 1445–58.Google Scholar
  25. Sambrook, J., Fritsch, E.R. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  26. Shapiro, J.T., Leng, M. and Felsenfeld, G. (1969) Deoxyribonucleic acid-polylysine complexes structure and nucleotide specificity.Biochem. J. 8, 3219–32.Google Scholar
  27. Velander, W.H., Page, R.L., Morcol, T., Russell, C.G., Canseco, R., Young, J.M., Drohan, W.N., Gwazdauskas, F.G., Wilkins, T.D. and Johnson, J.L. (1992a) Production of biologically active protein C in the milk of transgenic mice.Ann. NY Acad. Sci. 665, 391–403.Google Scholar
  28. Velander, W.H., Johnson, J.L., Page, R.L., Russell, C.G., Subramanian, A., Wilkins, T.D., Gwazdauskas, F.G., Pittius, C. and Drohan, W.N. (1992b) High level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C.Proc. Natl Acad. Sci USA 89, 12003–7.Google Scholar
  29. Wagner, E., Zenke, M., Cotten, M., Beug, H. and Birnstiel, M.L. (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells.Proc. Natl. Acad. Sci. USA 87, 3410–14.Google Scholar
  30. Wall R.J., Pursel, V.G., Hammer, R.E. and Brinster, R.L. (1985) Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei.Biol. Reprod. 32, 645–51.Google Scholar
  31. Wall, R.J., Pursel, V.G., Shamay, A.V., McKnight, R.A., Pittius, C.W. and Hennighausen, L. (1991) High-level synthesis of a heterologous milk protien in the mammary glands of transgenic swine.Proc. Natl Acad. Sci. USA 88, 1696–700.Google Scholar
  32. Widom, J. and Baldwin, R.L. (1980) Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6.J. Mol. Biol. 144, 431–53.Google Scholar
  33. Wright, G., Carver, A., Cottom, D., Reeves, D., Scott, A., Simons, P., Wilmut, I., Garner, I. and Colman, A. (1991) High, level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep.Bio/Technology 9, 830–4.Google Scholar
  34. Wu, G.Y. and Wu, C.H. (1987) Receptor-mediatedin vitro gene transformation by a soluble DNA carrier system.J. Biol. Chem. 262, 4429–32.Google Scholar
  35. Wu, G.Y. and Wu, C.H. (1988a) Evidence for targeted gene delivery to hep G hepatoma cellsin vitro.Biochem. J. 27, 887–92.Google Scholar
  36. Wu, G.Y. and Wu, C.H. (1988b) Receptor-mediated gene delivery and expressionin vitro.J. Biol. Chem. 263, 14621–4.Google Scholar

Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • Raymond L. Page
    • 1
  • Stephen P. Butler
    • 1
  • Anuradha Subramanian
    • 1
  • Francis C. Gwazdauskas
    • 2
  • John L. Johnson
    • 3
  • William H. Velander
    • 1
  1. 1.Department of Chemical EnginneringVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  2. 2.Department of Dairy ScienceVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  3. 3.Department of Biochemistry and Anaerobic MicrobiologyVirginia Polytechnic Institute and State UniversityBlacksburgUSA

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