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. 1985 Mar;37(2):295–310.

Transfer of human and murine globin-gene sequences into transgenic mice.

R K Humphries, P Berg, J DiPietro, S Bernstein, A Baur, A W Nienhuis, W F Anderson
PMCID: PMC1684563  PMID: 2580434

Abstract

We have studied the transfer of human and murine globin gene sequences into fertilized mouse oocytes by microinjection. Germline transmission was demonstrated for the human delta- and beta-globin genes contained in the bacteriophage lambda H beta G1. Expression of these human globin-gene sequences was not detectable in either erythroid or nonerythroid tissues. A recombinant plasmid containing the murine beta maj promoter region coupled to the prokaryotic coding sequence for galactokinase was also successfully transferred to two mice, and stable germline transmission of integrated DNA was demonstrated for at least 3 generations. Despite the presence of a murine globin-promoter sequence, expression of the mouse beta maj galactokinase fusion gene was not observed in primary or secondary animals in erythroid or nonerythroid tissues. Analysis of primary and secondary animals from both series of injections revealed extensive de novo methylation in the integrated microinjected DNA. Administration of 5-azacytidine to mice containing the mouse beta maj-promoted galactokinase gene resulted in partial hypomethylation was associated with an apparent two- to threefold increase in galactokinase (gal K) gene expression.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benz E. J., Geist C. E., Steggles A. W., Barker J. E., Nienhuis A. W. Hemoglobin switching in sheep and goats. Preparation and characterization of complementary DNAs specific for the alpha-, beta-, and gamma-globin messenger RNAs of sheep. J Biol Chem. 1977 Mar 25;252(6):1908–1916. [PubMed] [Google Scholar]
  2. Berg P. E., Yu J. K., Popovic Z., Schumperli D., Johansen H., Rosenberg M., Anderson W. F. Differential activation of the mouse beta-globin promoter by enhancers. Mol Cell Biol. 1983 Jul;3(7):1246–1254. doi: 10.1128/mcb.3.7.1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brinster R. L., Chen H. Y., Trumbauer M., Senear A. W., Warren R., Palmiter R. D. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell. 1981 Nov;27(1 Pt 2):223–231. doi: 10.1016/0092-8674(81)90376-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brinster R. L., Ritchie K. A., Hammer R. E., O'Brien R. L., Arp B., Storb U. Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice. Nature. 1983 Nov 24;306(5941):332–336. doi: 10.1038/306332a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chao M. V., Mellon P., Charnay P., Maniatis T., Axel R. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells. Cell. 1983 Feb;32(2):483–493. doi: 10.1016/0092-8674(83)90468-3. [DOI] [PubMed] [Google Scholar]
  6. Costantini F., Lacy E. Introduction of a rabbit beta-globin gene into the mouse germ line. Nature. 1981 Nov 5;294(5836):92–94. doi: 10.1038/294092a0. [DOI] [PubMed] [Google Scholar]
  7. Creusot F., Acs G., Christman J. K. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine. J Biol Chem. 1982 Feb 25;257(4):2041–2048. [PubMed] [Google Scholar]
  8. DeSimone J., Heller P., Hall L., Zwiers D. 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4428–4431. doi: 10.1073/pnas.79.14.4428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  10. Gordon J. W., Ruddle F. H. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science. 1981 Dec 11;214(4526):1244–1246. doi: 10.1126/science.6272397. [DOI] [PubMed] [Google Scholar]
  11. Gordon J. W., Scangos G. A., Plotkin D. J., Barbosa J. A., Ruddle F. H. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7380–7384. doi: 10.1073/pnas.77.12.7380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoppe P. C., Pitts S. Fertilization in vitro and development of mouse ova. Biol Reprod. 1973 May;8(4):420–426. doi: 10.1093/biolreprod/8.4.420. [DOI] [PubMed] [Google Scholar]
  13. Jähner D., Stuhlmann H., Stewart C. L., Harbers K., Löhler J., Simon I., Jaenisch R. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature. 1982 Aug 12;298(5875):623–628. doi: 10.1038/298623a0. [DOI] [PubMed] [Google Scholar]
  14. Kantor J. A., Turner P. H., Nienhuis A. W. Beta Thalassemia: mutations which affect processing of the beta-Globin mRNA precursor. Cell. 1980 Aug;21(1):149–157. doi: 10.1016/0092-8674(80)90122-1. [DOI] [PubMed] [Google Scholar]
  15. Lacy E., Roberts S., Evans E. P., Burtenshaw M. D., Costantini F. D. A foreign beta-globin gene in transgenic mice: integration at abnormal chromosomal positions and expression in inappropriate tissues. Cell. 1983 Sep;34(2):343–358. doi: 10.1016/0092-8674(83)90369-0. [DOI] [PubMed] [Google Scholar]
  16. Lawn R. M., Fritsch E. F., Parker R. C., Blake G., Maniatis T. The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA. Cell. 1978 Dec;15(4):1157–1174. doi: 10.1016/0092-8674(78)90043-0. [DOI] [PubMed] [Google Scholar]
  17. Ley T. J., Anagnou N. P., Noguchi C. T., Schechter A. N., DeSimone J., Heller P., Nienhuis A. W. DNA methylation and globin gene expression in patients treated with 5-azacytidine. Prog Clin Biol Res. 1983;134:457–474. [PubMed] [Google Scholar]
  18. Ley T. J., DeSimone J., Anagnou N. P., Keller G. H., Humphries R. K., Turner P. H., Young N. S., Keller P., Nienhuis A. W. 5-azacytidine selectively increases gamma-globin synthesis in a patient with beta+ thalassemia. N Engl J Med. 1982 Dec 9;307(24):1469–1475. doi: 10.1056/NEJM198212093072401. [DOI] [PubMed] [Google Scholar]
  19. Palmiter R. D., Brinster R. L., Hammer R. E., Trumbauer M. E., Rosenfeld M. G., Birnberg N. C., Evans R. M. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature. 1982 Dec 16;300(5893):611–615. doi: 10.1038/300611a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Palmiter R. D., Chen H. Y., Brinster R. L. Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell. 1982 Jun;29(2):701–710. doi: 10.1016/0092-8674(82)90186-6. [DOI] [PubMed] [Google Scholar]
  21. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  22. Sheffery M., Rifkind R. A., Marks P. A. Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1180–1184. doi: 10.1073/pnas.79.4.1180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Skow L. C., Burkhart B. A., Johnson F. M., Popp R. A., Popp D. M., Goldberg S. Z., Anderson W. F., Barnett L. B., Lewis S. E. A mouse model for beta-thalassemia. Cell. 1983 Oct;34(3):1043–1052. doi: 10.1016/0092-8674(83)90562-7. [DOI] [PubMed] [Google Scholar]
  24. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  25. Steward T. A., Wagner E. F., Mintz B. Human beta-globin gene sequences injected into mouse eggs, retained in adults, and transmitted to progeny. Science. 1982 Sep 10;217(4564):1046–1048. doi: 10.1126/science.6287575. [DOI] [PubMed] [Google Scholar]
  26. Wagner E. F., Stewart T. A., Mintz B. The human beta-globin gene and a functional viral thymidine kinase gene in developing mice. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5016–5020. doi: 10.1073/pnas.78.8.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wagner T. E., Hoppe P. C., Jollick J. D., Scholl D. R., Hodinka R. L., Gault J. B. Microinjection of a rabbit beta-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6376–6380. doi: 10.1073/pnas.78.10.6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Willing M. C., Nienhuis A. W., Anderson W. F. Selective activation of human beta-but not gamma-globin gene in human fibroblast x mouse erythroleukaemia cell hybrids. Nature. 1979 Feb 15;277(5697):534–538. doi: 10.1038/277534a0. [DOI] [PubMed] [Google Scholar]

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