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production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs

Abstract

Transgenic technologies that enable rapid movement between genotype and phenotype through specific loss-of-function, overexpression, or misexpression phenotypes will be crucial in the elucidation of gene sequences emerging from genome projects. This article describes detailed procedures for the generation of transgenic mice and rats by the injection of cloned DNA into the pronuclei of fertilized one-cell eggs.

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References

  1. Dunham, I., Shimizu, N., Roe, B. A., Chissoe, S., Hunt, A. R., Collins, J. E., et al. (1999) The DNA sequence of human chromosome 22. Nature 402, 489–495.

    PubMed  Article  CAS  Google Scholar 

  2. Murphy, D. and Carter, D. (1992) Transgenic approaches to modifying cell and tissue function. Curr. Opin. Cell Biol. 4, 274–279.

    PubMed  Article  CAS  Google Scholar 

  3. Constantini, F. and Lacy, E. (1981) Introduction of rabbit β-globin gene into the mouse germ line. Nature 294, 92–94.

    Article  Google Scholar 

  4. Harbers, K., Jahner, D., and Jaenisch, R. (1981) Microinjection of cloned retroviral genomes into the mouse zygotes: integration and expression in the animal. Nature 293, 540–542.

    PubMed  Article  CAS  Google Scholar 

  5. Brinster, R. L., Chen, H.Y., and Trumbauer, M. (1981) Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223–231.

    PubMed  Article  CAS  Google Scholar 

  6. Brinster, R. L., Chen, H.Y., Warren, R., Sarthy, A., Palmiter, R.D. (1982) Regulation of metallothioneinthymidine kinase fusion plasmids injected into mouse eggs. Nature 296, 39–42.

    PubMed  Article  CAS  Google Scholar 

  7. Kornberg, R. D. (1996) RNA polymerase II: transcription control. Trends in Biochemical Science 21, 325,326.

    Article  CAS  Google Scholar 

  8. Nikolov, D. B., Chen, H., Halay, E. D., Hoffman, A., Roeder, R. G., and Burley, S. K. (1996) Crystal structure of a human TATA box-binding protein/TATA element complex. Proc. Natl. Acad. Sci. USA 93, 4862–4867.

    PubMed  Article  CAS  Google Scholar 

  9. Li, X.-Y., Virbasius, A., Zhu, X., and Green, M. R. (1999) Enhancement of TBP binding by activators and general transcription factors. Nature 399, 605–609.

    PubMed  Article  CAS  Google Scholar 

  10. Roeder, R. G. (1996) The role of general initiation factor in transcription by RNA polymerase II. Trends in Biochemical Science 21, 327–334.

    Article  CAS  Google Scholar 

  11. Bjorklund, S. and Kim, Y-J. (1996) Mediator of transcription regulation. Trends in Biochemical Science 21, 335–337.

    Article  CAS  Google Scholar 

  12. Verrijzer, C. P. and Tijan, R. (1996) TAFs mediate transcriptional activation and promoter selectivity. Trends in Biochemical Sciences 21, 338–342.

    PubMed  Article  CAS  Google Scholar 

  13. Banerji, J., Olson, L., and Schaffner, W. (1983) A lymphocytes-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33, 729–740.

    PubMed  Article  CAS  Google Scholar 

  14. Queen, C. and Baltimore, D. (1983) Immunoglobulin gene transcription is activated by downstream elements. Cell 33, 741–748.

    PubMed  Article  CAS  Google Scholar 

  15. Gillies, S. D., Morrison, S.L., Oi, V.T., and Tonegawa, S. (1983) A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33, 717–728.

    PubMed  Article  CAS  Google Scholar 

  16. Walker, M. D., Edlund, T., Boulet, A. M., and Rutter, W. J. (1983) Cell-specific expression controlled by the 5′-flanking region of insulin and chymotrypsin. Nature 306, 557–561.

    PubMed  Article  CAS  Google Scholar 

  17. Ornitz, D. M., Palmiter, R. D., Hammer, R. E., Brinster, R. L., Swift, G. H., and MacDonald, R. J. (1985) Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice. Nature 313, 600–602.

    PubMed  Article  CAS  Google Scholar 

  18. Selden, R. F., Yun, J. S., Moore, D. D., Rowe, M. E., Malia, M. A., Wagner, T. E., et al. (1989) Glucocorticoid regulation of human growth hormone expression in transgenic mice amnd transiently transfected cells. J. Endocrinol. 122, 49–60.

    PubMed  CAS  Article  Google Scholar 

  19. Banerjee, S. A., Roffler-Tarlov, S., Szabo, M., Frohman, L., and Chikaraishi, D.M. (1994) DNA regulatory sequences of the rat tyrosine hydroxylase gene direct correct catecholoaminergic cell-type specificity of a human growth hormone reporter in the CNS of transgenic mice causing a dwarf phenotype. Mol. Brain Res. 24, 89–106.

    PubMed  Article  CAS  Google Scholar 

  20. Swanson, L. W., Simmons, D. M., Arriza, J., Hammer, R., Brinster, R., Rosenfeld, M. G., et al. (1985) Novel developmental specificity in the nervous system of transgenic animals expressing growth hormone fusion genes. Nature 317, 363–366.

    PubMed  Article  CAS  Google Scholar 

  21. Lacy, E., Roberts, S., Evans, E. P., Burtenshaw, M. D., and Costantini, F. D. (1983) A foreign β-globin gene in transgenic mice: Integration at abnormal chromosomal positions and expression in inappropriate tissues. Cell 34, 343–358.

    PubMed  Article  CAS  Google Scholar 

  22. Al-Shawi, R., Kinnaird, J., Burke, J., and Bishop, J. O. (1990) Expression of a foreign gene in a line of transgenic mice is modulated by chromosomal position effect. Mol. Cell. Biol. 10, 1192–1198.

    PubMed  CAS  Google Scholar 

  23. Huber, M. C., Bosch, F. X., Sippel, A. E., and Bonifer, C. (1994) Chromosomal position effect in chicken lysozyme gene transgenic mice are correlated with supression of DNase I hypersensitive site formation. Nucleic Acids Res. 22, 4195–4201.

    PubMed  Article  CAS  Google Scholar 

  24. Grosveld, F., van Assendelft, G. B., Greaves, D. R., and Kollias, G. (1987) Position-independent, high-level expression of human β-globin gene in transgenic mice. Cell 51, 975–985.

    PubMed  Article  CAS  Google Scholar 

  25. Bonifer, C., Hecht, A., Saueressig, H., Winter, H., Winter, D. M., and Sippel, A. E. (1991) Dynamic chromatin: the regulatory domin organization of eukaryotic gene loci. J. Cell. Biochem 47, 99–108.

    PubMed  Article  CAS  Google Scholar 

  26. Grosveld, F. (1999) Activation by locus control regions? Cur. Op. Genetics and Development. 9, 152–157.

    Article  CAS  Google Scholar 

  27. Stief, A., Winter, D. M., Strätling, W. H., and Sippel, A. E. (1989) A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341, 343–345.

    PubMed  Article  CAS  Google Scholar 

  28. Higgs, D. R., Wood, W. G., Jarman, A. P., Sharpe, J., Lida, J., Pretorius, I.-M., et al. (1990) A major positive regulatory region located far upstream of the human α-globin gene locus. Genes and Dev. 4, 1588–1601.

    PubMed  Article  CAS  Google Scholar 

  29. Jones, B. K., Monks, B. R., Liebhaber, S. A., and Cooke, N. E. (1995) The human growth hormone gene is regulated by multicomponent locus control region. Mol. Cell. Biol. 15, 7010–7021.

    PubMed  CAS  Google Scholar 

  30. Fraser, P., Hurst, J., Collis, P., and Grosveld, F. (1990) DNase I hypersensitive sites 1, 2 and 3 of the human β-globin dominant control region direct position-independent expression. Nucleic Acids Res. 18, 3503–3508.

    PubMed  Article  CAS  Google Scholar 

  31. Chung, J. H., Whiteley, M., and Felsenfeld, G. (1993) A 5′ element of the chicken β-globin domain serves as an insulator in human erythroid cells and protects against position effect in drosophila. Cell 74, 505–514.

    PubMed  Article  CAS  Google Scholar 

  32. Aronow, B. J., Ebert, C. A., Valerius, M. T., Potter, S. S., Wiginton, D. A., Witte, D. P., et al. (1995) Dissecting a locus control region: facilitation of enhancer function by extended enhancer-flanking sequences. Mol. Cell. Biol. 15, 1123–1135.

    PubMed  CAS  Google Scholar 

  33. Forrester, W. C., Epner, E., Driscoll, M.C., Enver, T., Brice, M., Papayannopoulou, T., et al. (1990) A deletion of the human β-globin locus activation region causes a major alteration in chromatin structure and replication across the entire β-globin locus. Genes and Dev. 4, 1637–1649.

    PubMed  Article  CAS  Google Scholar 

  34. Kioussis, D. and Festenstein, R. (1997) Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Current Biology 7, 614–619.

    CAS  Google Scholar 

  35. Wahle, E. and Keller, W. (1996) The biochemistry of polyadenylation. Trends in Biochemical Science 21, 247–250.

    Article  CAS  Google Scholar 

  36. Shilatifard, A., Conaway, J. W., and Conaway, R. C. (1997) Mechanism and regulation of transcriptional elongation and termination by RNA polymerase II. Cur. Op. Genetics and Development 7, 199–204.

    Article  CAS  Google Scholar 

  37. Decker, C. J. and Parker, R. (1994) Mechanisms of mRNA degradation in eukaryotes. Trends in Biochemical Sciences 19, 336–340.

    PubMed  Article  CAS  Google Scholar 

  38. Sachs, A. B. (1993) Messenger RNA degradation in eukaryotes. Cell 74, 413–421.

    PubMed  Article  CAS  Google Scholar 

  39. Kozak, M. (1985) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292.

    Article  Google Scholar 

  40. Palmiter, R. D., Brinster, R. L., Hammer, R. E., Trumbauer, M. E., Rosenfeld, M. G., Birnberg, N. C., et al. (1982) Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300, 611–615.

    PubMed  Article  CAS  Google Scholar 

  41. McGrane, M. M., de Vente, J., Yun, J., Bloom, J., Park, E., Wynshaw-Boris, A., et al. (1988) Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/ bovine growth hormone gene in transgenic mice. J. Biol. Chem 263, 11,443–11,451.

    CAS  Google Scholar 

  42. Russo, A. F., Crenshaw, E. B, Lira, S. A., Simmons, D. M., Swanson, L.W., and Rosenfeld, M. G. (1988) Neuronal expression of chimeric genes in transgenic mice. Neuron 1, 311–320.

    PubMed  Article  CAS  Google Scholar 

  43. Hollingshead, P. G., Martin, L., Pitts, S.L., and Stewart, T.A. (1989) A dominant phenocopy of hypopituitarism in transgenic mice resulting from central nervous system synthesis of human growth hormone. Endocrinol. 125, 1556–1564.

    CAS  Article  Google Scholar 

  44. Rossant, J., Zirngibl, R., Cado, D., Shago, M., and Giguére, V. (1991) Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes and Dev. 5, 1333–1344.

    PubMed  Article  CAS  Google Scholar 

  45. Smeyne, R. J., Schilling, K., Robertson, L., Luk, D., Oberdick, J., Curran, T., et al. (1992) Fos-lacZ transgenic mice: Mapping sites of gene induction in the central nervous system. Neuron 8, 13–23.

    PubMed  Article  CAS  Google Scholar 

  46. Ross, S. R., Solter, D. (1985) Glucocorticoid regulation of mouse mammary tumor virus sequences in transgenic mice. Proc. Natl. Acad. Sci. USA 83, 5880–5884.

    Article  Google Scholar 

  47. Morello, D., Moore, G., Salmon, A. M., Yaniv, M., and Babinet, C. (1986) Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice. EMBO J. 5, 1877–1883.

    PubMed  CAS  Google Scholar 

  48. Chen, W. Y., Wight, D. C., Wagner, T. E., and Kopchick, J. J. (1990) Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice. Proc. Natl. Acad. Sci. USA 87, 5061–5065.

    PubMed  Article  CAS  Google Scholar 

  49. Schinke, M., Baltatu, O., Bohm, M., Peters, J., Rascher, W., Bricca, G., et al. (1999) Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen. Proc. Natl. Acad. Sci. USA 96, 3975–3980.

    PubMed  Article  CAS  Google Scholar 

  50. Piccioli, P., Di Luzio, A., Amann, R., Schuligoi, R., Surani, M. A., Donnerer, J., et al. (1995) Neuroantibodies: ectopic expression of a recombinant anti-substance P antibody in the central nervous system of transgenic mice. Neuron 15, 373–384.

    PubMed  Article  CAS  Google Scholar 

  51. Behringer, R. R. L. S., Mathews, R. D., Palmiter, R. D., and Brinster, R. L. (1988) Dwarf mice produced by genetic ablation of growth hormone expressing cells. Genes and Dev. 2, 453–461.

    PubMed  Article  CAS  Google Scholar 

  52. Struthers, R. S., Vale, W. W., Arias, C., Sawchenko, P. E., and Montminy, M. R. (1991) Somatotroph hypoplasia and dwarfism in transgenic mice expressing a nonphosphorylatable CREB mutant. Nature 350, 622–624.

    PubMed  Article  CAS  Google Scholar 

  53. Burton, F. H., Hasel, K. W., Bloom, F. E., and Sutcliffe, J. G. (1991) Pituitary hyperplasia and giantism in mice caused by cholera toxin transgene. Nature 350, 74–77.

    PubMed  Article  CAS  Google Scholar 

  54. Flavell, D. M., Wells, T., Wells, S. E., Carmignac, D. F., Thomas, G. B., and Robinson, I. C. (1996) Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons. EMBO J. 15, 3871–3879.

    PubMed  CAS  Google Scholar 

  55. Zeng, Q., Carter, D. A., and Murphy, D. (1994) Cell specific expression of a vasopressin transgene in rats. J. Neuroendocrinol. 6, 469–477.

    PubMed  Article  CAS  Google Scholar 

  56. (1985) NIH Guide for the Care and Use of Laboratory Animals National Institutes of Health publication No 85-23, Washington, DC.

  57. Brinster, R. L., Chen, H. Y., Trunbauer, M. E., Yagle, M. K., and Palmiter, R. D. (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438–4442.

    PubMed  Article  CAS  Google Scholar 

  58. Mullins, J. J., Peters, J., and Ganten, D. (1990) Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature 344, 541–544.

    PubMed  Article  CAS  Google Scholar 

  59. Hogan, B., Constantini, F., and Lacy, E. (ed.) (1986) Manipulating the Mouse Embryo—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  60. Chada, K., Magram, J., Raphael, K., Radice, G., Lacy, E., and Constantini, F. (1985) Specific expression of a foreign B-globin gene in erythroid cells in transgenic mice. Nature 314, 377–380.

    PubMed  Article  CAS  Google Scholar 

  61. Oberdick, J., Smeyne, R. J., Mann, J. R., Zackson, S., and Morgan, J. I. (1990) The promoter that drives transgene expression in cerebellar Purkinje and retinal bipolar neurons. Science 248, 223–236.

    PubMed  Article  CAS  Google Scholar 

  62. Brinster, R. L., Allen, J. M., Behringer, R. R., Gelinas, R. E., and Palmiter, R. D. (1988) Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. USA 85, 836–840.

    PubMed  Article  CAS  Google Scholar 

  63. Young, W. S. III, Reynolds, K., Shepard, E. A., Gainer, H., and Castel, M. (1990) Cell specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol. 2, 917.

    Article  CAS  PubMed  Google Scholar 

  64. Schedl, A., Montoliu, L., Kelsey, G., and Schutz, G. (1993) A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature 362, 258–261.

    PubMed  Article  CAS  Google Scholar 

  65. Swanson, L. W., Simmons, D. M., Azzira, J., Hammer, R., and Brinster, R. (1985) Novel developmental specificity in the nervous system of transgenic animal expressing growth hormone fusion gene. Nature 317, 363–366.

    PubMed  Article  CAS  Google Scholar 

  66. Vogelstein, B. and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 82, 615–619.

    Article  Google Scholar 

  67. Constantini, F. and Lacy, E. (1981) Introduction of a rabbit B-globin gene into the mouse germ line. Nature 294, 92–94.

    Article  Google Scholar 

  68. Page, R. L., Butler, S. P., Subramaniam, A., Gwazdauskas, F. C., Johnson, J. L., and Velander, W. H., (1995) Transgenesis in mice by cytoplasmic injection of polylysinetDNA mixtures. Transgenic Res. 4, 353–360.

    PubMed  Article  CAS  Google Scholar 

  69. Whittingham, D. G. (1971) Culture of mouse ova. J. Reprod. Fertil. Suppl. 14, 7–21.

    PubMed  CAS  Google Scholar 

  70. Pomeroy, K. O. (1991) Cryopreservation of transgenic mice. GATA 8, 95–101.

    CAS  Google Scholar 

  71. Kasai, M., Komi, J. H., Takakomo, A., Tsudura, H., Sakurai, T., and Machada, T. (1990) A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil. 89, 91–97.

    PubMed  CAS  Article  Google Scholar 

  72. Kono, T., Suzuki, O., and Tsunoda, Y. (1988) Cryopreservation of rat blastocysts by vitrification. Cryobiology 25, 170–173.

    PubMed  Article  CAS  Google Scholar 

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Si-Hoe, S.L., Wells, S. & Murphy, D. production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs. Mol Biotechnol 17, 151–182 (2001). https://doi.org/10.1385/MB:17:2:151

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Index Entries

  • Transgenic rats
  • transgenic mice
  • microinjection
  • superovulation
  • oviduct transfer
  • in vitro fertilization
  • cryopreservation