Inducible Transgene Expression in Mouse Stem Cells

  • David T. Ting
  • Michael Kyba
  • George Q. Daley
Part of the Methods in Molecular Medicine book series (MIMM, volume 105)


Embryonic stem (ES) cells serve as a potentially unlimited source of cells and tissues to treat a number of genetic and malignant diseases. The differentiation of these cells into specific cell types is an area of very active investigation. One method of manipulating ES cell differentiation is through the alteration of gene expression. There are a multitude of different methods for expressing a target gene in ES cells, but most are limited in their ability to provide spatial, temporal, and quantitative control of gene expression. These properties are important because many developmentally interesting genes are regulated in at least one of these ways. This chapter will address these limitations through the use of an ES cell line with a doxycycline-inducible transgene system. A characterization of this inducible transgene system will be discussed, as well as the use of this system to develop ES-derived long-term engrafting hematopoietic stem cells. This demonstration is one of many possible uses for this powerful and versatile system.

Key Words

Embryonic stem (ES) cell genetic modification inducible transgene tet operon rtTA hematopoiesis 


  1. 1.
    Evans, M. J. and Kaufman, M. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.PubMedCrossRefGoogle Scholar
  2. 2.
    Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638.PubMedCrossRefGoogle Scholar
  3. 3.
    Robertson, E., Bradley, A., Kuehn, M., and Evans, M. (1986) Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445–448.PubMedCrossRefGoogle Scholar
  4. 4.
    Kuehn, M. R., Bradley, A., Robertson, E. J., and Evans, M. J. (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326, 295–298.PubMedCrossRefGoogle Scholar
  5. 5.
    Cherry, S. R., Biniszkiewicz, D., van Parijs, L., Baltimore, D., and Jaenisch, R. (2000) Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol. Cell Biol. 20, 7419–7426.PubMedCrossRefGoogle Scholar
  6. 6.
    Robbins, P. B., Yu, X. J., Skelton, D. M., Pepper, K. A., Wasserman, R. M., Zhu, L., et al. (1997) Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells. J. Virol. 71, 9466–9474.PubMedGoogle Scholar
  7. 7.
    Hildinger, M., Eckert, H. G., Schilz, A. J., John, J., Ostertag, W., and Baum, C. (1998) FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells. Gene Ther. 5, 1575–1579.PubMedCrossRefGoogle Scholar
  8. 8.
    Ketteler, R., Glaser, S., Sandra, O., Martens, U. M., and Klingmuller, U. (2002) Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther. 9, 477–487.PubMedCrossRefGoogle Scholar
  9. 9.
    Hamaguchi, I., Woods, N. B., Panagopoulos, I., Andersson, E., Mikkola, H., Fahlman, C., et al. (2000) Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro. J. Virol. 74, 10,778–10,784.PubMedCrossRefGoogle Scholar
  10. 10.
    Kafri, T., van Praag, H., Gage, F. H., and Verma, I. M. (2000) Lentiviral vectors: regulated gene expression. Mol. Ther. 1, 516–521.PubMedCrossRefGoogle Scholar
  11. 11.
    Ramezani, A., Hawley, T. S., and Hawley, R. G. (2000) Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol. Ther. 2, 458–469.PubMedCrossRefGoogle Scholar
  12. 12.
    Pfeifer, A., Ikawa, M., Dayn, Y., and Verma, I. M. (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA 99, 2140–2145.PubMedCrossRefGoogle Scholar
  13. 13.
    Vigna, E., Cavalieri, S., Ailles, L., Geuna, M., Loew, R., Bujard, H., and Naldini, L. (2002) Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol. Ther. 5, 252–261.PubMedCrossRefGoogle Scholar
  14. 14.
    Jin, L., Siritanaratkul, N., Emery, D. W., Richard, R. E., Kaushansky, K., Papayannopoulou, T., et al. (1998) Targeted expansion of genetically modified bone marrow cells. Proc. Natl. Acad. Sci. USA 95, 8093–8097.PubMedCrossRefGoogle Scholar
  15. 15.
    Jin, L., Zeng, H., Chien, S., Otto, K. G., Richard, R. E., Emery, D. W., and Blau, C. A. (2000) In vivo selection using a cell-growth switch. Nat. Genet. 26, 64–66.PubMedCrossRefGoogle Scholar
  16. 16.
    Hofmann, A., Nolan, G. P., and Blau, H. M. (1996) Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc. Natl. Acad. Sci. USA 93, 5185–5190.PubMedCrossRefGoogle Scholar
  17. 17.
    Doetschman, T., Gregg, R. G., Maeda, N., Hooper, M. L., Melton, D. W., Thompson, S., et al. (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578.PubMedCrossRefGoogle Scholar
  18. 18.
    Thomas, K. R. and Capecchi, M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.PubMedCrossRefGoogle Scholar
  19. 19.
    Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352.PubMedCrossRefGoogle Scholar
  20. 20.
    Schwartzberg, P. L., Goff, S. P., and Robertson, E. J. (1989) Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246, 799–803.PubMedCrossRefGoogle Scholar
  21. 21.
    Hasty, P., Ramirez-Solis, R., Krumlauf, R., and Bradley, A. (1991) Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature 350, 243–246.PubMedCrossRefGoogle Scholar
  22. 22.
    Valancius, V. and Smithies, O. (1991) Testing an “in-out” targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol. Cell Biol. 11, 1402–1408.PubMedGoogle Scholar
  23. 23.
    Rudolph, U., Brabet, P., Hasty, P., Bradley, A., and Birnbaumer, L. (1993) Disruption of the G(i2) alpha locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair. Transgenic Res. 2, 345–355.PubMedCrossRefGoogle Scholar
  24. 24.
    Askew, G. R., Doetschman, T., and Lingrel, J. B. (1993) Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol. Cell Biol. 13, 4115–4124.PubMedGoogle Scholar
  25. 25.
    Stacey, A., Schnieke, A., McWhir, J., Cooper, J., Colman, A., and Melton, D. W. (1994) Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol. Cell Biol. 14, 1009–1116.PubMedGoogle Scholar
  26. 26.
    Detloff, P. J., Lewis, J., John, S. W., Shehee, W. R., Langenbach, R., Maeda, N., and Smithies, O. (1994) Deletion and replacement of the mouse adult beta-globin genes by a “plug and socket” repeated targeting strategy. Mol. Cell Biol. 14, 6936–6943.PubMedGoogle Scholar
  27. 27.
    Sauer, B. (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14, 381–392.PubMedCrossRefGoogle Scholar
  28. 28.
    Hadjantonakis, A. K., Pirity, M., and Nagy, A. (1999) Cre recombinase mediated alterations of the mouse genome using embryonic stem cells. Methods Mol. Biol. 97, 101–122.PubMedGoogle Scholar
  29. 29.
    Le, Y. and Sauer, B. (2000) Conditional gene knockout using cre recombinase. Methods Mol. Biol. 136, 477–485.PubMedGoogle Scholar
  30. 30.
    Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109.PubMedCrossRefGoogle Scholar
  31. 31.
    Zhang, Y., Riesterer, C., Ayrall, A. M., Sablitzky, F., Littlewood, T. D., and Reth, M. (1996) Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 24, 543–548.PubMedCrossRefGoogle Scholar
  32. 32.
    Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., and McMahon, A. P. (1998) Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8, 1323–1326.PubMedCrossRefGoogle Scholar
  33. 33.
    Verrou, C., Zhang, Y., Zurn, C., Schamel, W. W., and Reth, M. (1999) Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. J. Biol. Chem. 380, 1435–1438.Google Scholar
  34. 34.
    Indra, A. K., Warot, X., Brocard, J., Bornert, J. M., Xiao, J. H., Chambon, P., and Metzger, D. (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327.PubMedCrossRefGoogle Scholar
  35. 35.
    Chiba, H., Chambon, P., and Metzger, D. (2000) F9 embryonal carcinoma cells engineered for tamoxifen-dependent Cre-mediated site-directed mutagenesis and doxycycline-inducible gene expression. Exp. Cell Res. 260, 334–339.PubMedCrossRefGoogle Scholar
  36. 36.
    Fuhrmann-Benzakein, E., Garcia-Gabay, I., Pepper, M. S., Vassalli, J. D., and Herrera, P. L. (2000) Inducible and irreversible control of gene expression using a single transgene. Nucleic Acids Res. 28, E99.PubMedCrossRefGoogle Scholar
  37. 37.
    Imai, T., Jiang, M., Kastner, P., Chambon, P., and Metzger, D. (2001) Selective ablation of retinoid X receptor alpha in hepatocytes impairs their lifespan and regenerative capacity. Proc. Natl. Acad. Sci. USA 98, 4581–4586.PubMedCrossRefGoogle Scholar
  38. 38.
    Imai, T., Jiang, M., Chambon, P., and Metzger, D. (2001) Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes. Proc. Natl. Acad. Sci. USA 98, 224–228.PubMedGoogle Scholar
  39. 39.
    Endoh, M., Ogawa, M., Orkin, S., and Nishikawa, S.-I. (2002) SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation. EMBO J. 21, 6700–6708.PubMedCrossRefGoogle Scholar
  40. 40.
    Guo, C., Yang, W., and Lobe, C. G. (2002) A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18.PubMedCrossRefGoogle Scholar
  41. 41.
    Hayashi, S., and McMahon, A. P. (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244, 305–318.PubMedCrossRefGoogle Scholar
  42. 42.
    Petrich, B. G., Molkentin, J. D., and Wang, Y. (2003) Temporal activation of c-Jun N-terminal kinase in adult transgenic heart via cre-loxP-mediated DNA recombination. FASEB J. 19, 19.Google Scholar
  43. 43.
    Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551.PubMedCrossRefGoogle Scholar
  44. 44.
    Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769.PubMedCrossRefGoogle Scholar
  45. 45.
    Baron, U., Gossen, M., and Bujard, H. (1997) Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res. 25, 2723–2729.PubMedCrossRefGoogle Scholar
  46. 46.
    Niwa, H., Burdon, T., Chambers, I., and Smith, A. (1998) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–2060.PubMedCrossRefGoogle Scholar
  47. 47.
    Niwa, H., Miyazaki, J., and Smith, A. G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376.PubMedCrossRefGoogle Scholar
  48. 48.
    Era, T. and Witte, O. N. (2000) Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate. Proc. Natl. Acad. Sci. USA 97, 1737–1742.PubMedCrossRefGoogle Scholar
  49. 49.
    Furth, P. A., St Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. (1994) Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. USA 91, 9302–9306.PubMedCrossRefGoogle Scholar
  50. 50.
    Bjornsson, J. M., Andersson, E., Lundstrom, P., Larsson, N., Xu, X., Repetowska, E., Humphries, R. K., et al. (2001) Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10. Blood 98, 3301–3308.PubMedCrossRefGoogle Scholar
  51. 51.
    Schonig, K., Schwenk, F., Rajewsky, K., and Bujard, H. (2002) Stringent doxycycline dependent control of CRE recombinase in vivo. Nucleic Acids Res. 30, e134.PubMedCrossRefGoogle Scholar
  52. 52.
    Moody, S. E., Sarkisian, C. J., Hahn, K. T., Gunther, E. J., Pickup, S., Dugan, K. D., et al. (2002) Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis. Cancer Cell 2, 451–461.PubMedCrossRefGoogle Scholar
  53. 53.
    Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L., et al. (1995) Failure of blood-island formation and vasculogenesis in Flk-1 deficient mice. Nature 376, 62–66.PubMedCrossRefGoogle Scholar
  54. 54.
    Shivdasani, R. A., Mayer, E. L., and Orkin, S. H. (1995) Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373, 432–434.PubMedCrossRefGoogle Scholar
  55. 55.
    Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W., and Orkin, S. H. (1996) The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 47–57.PubMedCrossRefGoogle Scholar
  56. 56.
    Shalaby, F., Ho, J., Stanford, W. L., Fischer, K.-D., Schuh, A. C., Schwartz, L., et al. (1997) A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89, 981–990.PubMedCrossRefGoogle Scholar
  57. 57.
    North, T., Gu, T. L., Stacy, T., Wang, Q., Howard, L., Binder, M., et al. (1999) Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. Development 126, 2563–2575.PubMedGoogle Scholar
  58. 58.
    Mikkola, H. K. A., Klintman, J., Yang, H., Hock, H., Schlaeger, T. M., Fujiwara, Y., et al. (2003) Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene. Nature 421, 547–551.PubMedCrossRefGoogle Scholar
  59. 59.
    Cho, S. K., Bourdeau, A., Letarte, M., and Zuniga-Pflucker, J. C. (2001) Expression and function of CD105 during the onset of hematopoiesis from Flk1(+) precursors. Blood 98, 3635–3642.PubMedCrossRefGoogle Scholar
  60. 60.
    Lacaud, G., Gore, L., Kennedy, M., Kouskoff, V., Kingsley, P., Hogan, C., et al. (2002) Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 100, 458–468.PubMedCrossRefGoogle Scholar
  61. 61.
    Kyba, M., Perlingeiro, R. C., and Daley, G. Q. (2002) HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109, 29–37.PubMedCrossRefGoogle Scholar
  62. 62.
    Sauvageau, G., Landsdorp, P. M., Eaves, C. J., Hogge, D. E., Dragowska, W. H., Reid, D. S., et al. (1994) Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc. Natl. Acac. Sci. USA 91, 12,223–12,227.CrossRefGoogle Scholar
  63. 63.
    McGrath, K. E. and Palis, J. (1997) Expression of homeobox genes, including an insulin promoting factor, in the murine yolk sac at the time of hematopoietic initiation. Mol. Reprod. Dev. 48, 145–153.PubMedCrossRefGoogle Scholar
  64. 64.
    Onishi, M., Nosaka, T., Misawa, K., Mui, A. L.-F., Gorman, D., McMahon, M., et al. (1998) Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation. Mol. Cell Biol. 18, 3871–3879.PubMedGoogle Scholar
  65. 65.
    Moriggl, R., Topham, D. J., Teglund, S., Sexl, V., McKay, C., Wang, D., et al. (1999) Stat5 activation is uniquely associated with cytokine signaling in peripheral T cells. Immunity 10, 249–259.PubMedCrossRefGoogle Scholar
  66. 66.
    Socolovsky, M., Fallon, A. E. J., Wang, S., Brugnara, C., and Lodish, H. F. (1999) Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-XL induction. Cell 98, 181–191.PubMedCrossRefGoogle Scholar
  67. 67.
    Schwaller, J., Parganas, E., Wang, D., Cain, D., Aster, J. C., Williams, I. R., et al. (2000) Stat5 is essential for the myelo-and lymphoproliferative disease induced by TEL/JAK2. Mol. Cell 6, 693–704.PubMedCrossRefGoogle Scholar
  68. 68.
    Bradley, H. L., Hawley, T. S., and Bunting, K. D. (2002) Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells. Blood 100, 3983–3989.PubMedCrossRefGoogle Scholar
  69. 69.
    Bunting, K. D., Bradley, H. L., Hawley, T. S., Moriggi, R., Sorrentino, B. P., and Ihle, J. N. (2002) Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5. Blood 99, 479–487.PubMedCrossRefGoogle Scholar
  70. 70.
    Snow, J. W., Abraham, N., Ma, M. C., Abbey, N. W., Herndier, B., and Goldsmith, M. A. (2002) STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells. Blood 99, 95–101.PubMedCrossRefGoogle Scholar
  71. 71.
    Buitenhuis, M., Baltus, B., Lammers, J.-W. J., Coffer, P. J., and Koenderman, L. (2003) Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood-derived CD34+ cells. Blood 101, 134–142.PubMedCrossRefGoogle Scholar
  72. 72.
    Carlesso, N., Frank, D. A., and Griffin, J. D. (1996) Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J. Exp. Med. 183, 811–820.PubMedCrossRefGoogle Scholar
  73. 73.
    Frank, D. A. and Varticovski, L. (1996) BCR/abl leads to the constitutive activation of Stat proteins, and shares an epitope with tyrosine phosphorylated Stats. Leukemia 10, 1724–1730.PubMedGoogle Scholar
  74. 74.
    Ilaria, R. L. J. and Etten, R. A. V. (1996) P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J. Biol. Chem. 271, 31,704–31,710.PubMedCrossRefGoogle Scholar
  75. 75.
    Shuai, K., Halpern, J., Hoeve, J. T., Rao, X., and Sawyers, C. L. (1996) Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 13, 247–254.PubMedGoogle Scholar
  76. 76.
    Nieborowska-Skorska, M., Wasik, M. A., Slupianek, A., Salomoni, P., Kitamura, T., Calabretta, B., and Skorski, T. (1999) Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis. J. Exp. Med. 189, 1229–1242.PubMedCrossRefGoogle Scholar
  77. 77.
    Hoover, R. R., Gerlach, M. J., Koh, E. Y., and Daley, G. Q. (2001) Cooperative and redundant effects of STAT5 and Ras signaling in BCR/ABL transformed hematopoietic cells. Oncogene 20, 5826–5835.PubMedCrossRefGoogle Scholar
  78. 78.
    Robb, L., Elwood, N. J., Elefanty, A. G., Kontgen, F., Li, R., Barnett, L. D., and Begley, C. G. (1996) The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J. 15, 4123–4129.PubMedGoogle Scholar
  79. 79.
    Drake, C. J., Brandt, S. J., Trusk, T. C., and Little, C. D. (1997) TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis. Dev. Biol. 192, 1–30.CrossRefGoogle Scholar
  80. 80.
    Elefanty, A. G., Begley, C. G., Metcalf, D., Barnett, L., Kontgen, F., and Robb, L. (1998) Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ “knock-in”. Proc. Natl. Acad. Sci. USA 95, 11,897–11,902.PubMedCrossRefGoogle Scholar
  81. 81.
    Gering, M., Rodaway, A. R., Gottgens, B., Patient, R. K., and Green, A. R. (1998) The SCL gene specifies haemangioblast development from early mesoderm. EMBO J. 17, 4029–4045.PubMedCrossRefGoogle Scholar
  82. 82.
    Mead, P. E., Kelley, C. M., Hahn, P. S., Piedad, O., and Zon, L. I. (1998) SCL specifies hematopoietic mesoderm in Xenopus embryos. Development 125, 2611–2620.PubMedGoogle Scholar
  83. 83.
    Elefanty, A. G., Begley, C. G., Hartley, L., Papaevangeliou, B., and Robb, L. (1999) SCL expression in the mouse embryo detected with a targeted lacZ reporter gene demonstrates its localization to hematopoietic, vascular, and neural tissues. Blood 94, 3754–3763.PubMedGoogle Scholar
  84. 84.
    Robertson, S. M., Kennedy, M., Shannon, J. M., and Keller, G. (2000) A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1. Development 127, 2447–2459.PubMedGoogle Scholar
  85. 85.
    Mead, P. E., Deconinck, A. E., Huber, T. L., Orkin, S. H., and Zon, L. I. (2001) Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins. Development 128, 2301–2308.PubMedGoogle Scholar
  86. 86.
    Chung, Y. S., Zhang, W. J., Arentson, E., Kingsley, P. D., Palis, J., and Choi, K. (2002) Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression. Development 129, 5511–5520.PubMedCrossRefGoogle Scholar
  87. 87.
    Ema, M., Faloon, P., Zhang, W. J., Hirashima, M., Reid, T., Stanford, W. L., et al. (2003) Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Development 17, 380–393.Google Scholar
  88. 88.
    Hall, M. A., Curtis, D. J., Metcalf, D., Elefanty, A. G., Sourris, K., L, L. R., Gothert, J. R., et al. (2003) The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12. Proc. Natl. Acad. Sci. USA 100, 992–997.PubMedCrossRefGoogle Scholar
  89. 89.
    Nakano, T., Kodama, H., and Honjo, T. (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101.PubMedCrossRefGoogle Scholar
  90. 90.
    Wutz, A. and Jaenisch, R. (2000) A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695–705.PubMedCrossRefGoogle Scholar
  91. 91.
    Wutz, A., Rasmussen, T. P., and Jaenisch, R. (2002) Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat. Genet. 30, 167–174.PubMedCrossRefGoogle Scholar
  92. 92.
    Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A., Kerr, W. G., and Soriano, P. (1997) Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794.PubMedCrossRefGoogle Scholar
  93. 93.
    Baron, U., Freundlieb, S., Gossen, M., and Bujard, H. (1995) Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 23, 3605–3606.PubMedCrossRefGoogle Scholar
  94. 94.
    Kyba, M., Perlingeiro, R. C. R., and Daley, G. Q. (2003) Development of hematopoietic repopulating cells from embryonic stem cells, in Methods in Enzymology (Wassarman, P. M. and Keller, G. M., eds.), pp. 114–129.Google Scholar
  95. 95.
    Martin, G. R. and Evans, M. J. (1975) Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. USA 72, 1441–1445.PubMedCrossRefGoogle Scholar
  96. 96.
    Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol. Exp. Morphol. 87, 27–45.PubMedGoogle Scholar
  97. 97.
    Schmidt, R. M., Bruyns, E., and Snodgrass, H. R. (1991) Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 5, 718–740.Google Scholar
  98. 98.
    Spangrude, G. J., Heimfeld, S., and Weissman, I. L. (1988) Purification and Characterization of Mouse Hematopoietic Stem Cells. Science 241, 58–62.PubMedCrossRefGoogle Scholar
  99. 99.
    Uchida, N. and Weissman, I. L. (1992) Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin-Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J. Exp. Med. 175, 175–184.PubMedCrossRefGoogle Scholar
  100. 100.
    Jurecic, R., Van, N. T., and Belmont, J. W. (1993) Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from mice with an Ly-6a haplotype. Blood 82, 2673–2683.PubMedGoogle Scholar
  101. 101.
    Uchida, N., Aguila, H. L., Fleming, W. H., Jerabek, L., and Weissman, I. L. (1994) Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells. Blood 83, 3758–3779.PubMedGoogle Scholar
  102. 102.
    Morrison, S. J., Hemmati, H. D., Wandycz, A. M., and Weissman, I. L. (1995) The purification and characterization of fetal liver hematopoietic stem cells. Proc. Natl. Acac. Sci. USA 92, 10,302–10,306.CrossRefGoogle Scholar
  103. 103.
    Osawa, M., Nakamura, K., Nishi, N., Takahasi, N., Tokuomoto, Y., Inoue, H., and Nakauchi, H. (1996) In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/-) hemopoietic stem cells. J. Immunol. 156, 3207–3214.PubMedGoogle Scholar
  104. 104.
    Morrison, S. J., Wandycz, A. M., Hemmati, H. D., Wright, D. E., and Weissman, I. L. (1997) Identification of a lineage of multipotent hematopoietic progenitors. Development 124, 1929–1939.PubMedGoogle Scholar
  105. 105.
    Ma, X., Robin, C., Ottersbach, K., and Dzierzak, E. (2002) The Ly-6A (Sca-1) GFP transgene is expressed in all adult mouse hematopoietic stem cells. Stem Cells 20, 514–521.PubMedCrossRefGoogle Scholar
  106. 106.
    Hanson, P., Mathews, V., Marrus, S. H., and Graubert, T. A. (2003) Enhanced green fluorescent protein targeted to the Sca-1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo. Exp. Hematol. 31, 159–167.PubMedCrossRefGoogle Scholar
  107. 107.
    Ito, C. Y., Li, C. Y., Bernstein, A., Dick, J. E., and Stanford, W. L. (2003) Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice. Blood 101, 517–523.PubMedCrossRefGoogle Scholar
  108. 108.
    Mitjavila-Garcia, M. T., Cailleret, M., Godin, I., Nogueira, M. M., Cohen-Solal, K., Schiavon, V., et al. (2002) Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development 129, 2003–2013.PubMedGoogle Scholar
  109. 109.
    Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D., and Orkin, S. H. (2003) Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101, 508–516.PubMedCrossRefGoogle Scholar
  110. 110.
    Shockett, P., Difilippantonio, M., Hellman, N., and Schatz, D. G. (1995) A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc. Natl. Acad. Sci. USA 92, 6522–6526.PubMedCrossRefGoogle Scholar
  111. 111.
    Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., and Hillen, W. (2000) Exploring the equence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97, 7963–7968.PubMedCrossRefGoogle Scholar
  112. 112.
    Akagi, K., Kanai, M., Saya, H., Kozu, T., and Berns, A. (2001) A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain. Nucleic Acids Res. 29, E23.PubMedCrossRefGoogle Scholar
  113. 113.
    Chung, S., Andersson, T., Sonntag, K. C., Bjorklund, L., Isacson, O., and Kim, K. S. (2002) Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20, 139–145.PubMedCrossRefGoogle Scholar
  114. 114.
    Baron, U., Schnappinger, D., Helbl, V., Gossen, M., Hillen, W., and Bujard, H. (1999) Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc. Natl. Acad. Sci. USA 96, 1013–1018.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • David T. Ting
    • 1
  • Michael Kyba
    • 2
  • George Q. Daley
    • 3
  1. 1.Harvard-M.I.T. Program in Health Sciences and TechnologyHarvard Medical SchoolBoston
  2. 2.Center for Developmental BiologyUT Southwestern Medical CenterDallas
  3. 3.Division of Hematology/OncologyChildren’s HospitalBoston

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