Retroviral Integration Site Analysis in Hematopoietic Stem Cells

  • Olga S. Kustikova
  • Christopher Baum
  • Boris Fehse
Part of the Methods in Molecular Biology™ book series (MIMB, volume 430)


Stable transgene insertion into a host genome irrevocably and unambiguously marks individual cells and all their descendants, i.e., the respective cell clone. Based thereon, retroviral gene marking has become an important tool for investigating the in vivo fate of different cell types, both in animal models and in clinical gene transfer. Moreover, identification of (vector) insertion sites in malignant clones transformed because of insertional activation of proto-oncogenes after experimental as well as therapeutic retroviral gene transfer has resulted in new insights into oncogenic transformation of hematopoietic stem cells (HSCs). However, because of the high sensitivity of the PCR-based methods for insertion site detection, researchers are often confronted with large numbers of different insertion sites/cell clones whose contribution to the given state is hard to judge. A relatively simple ligation-mediated polymerase chain reaction (LM-PCR) method allows the preferential analysis of insertion sites in those cell clones that significantly contributed to the cell pool analyzed. In murine bone marrow transplantation models, we have shown that this method is very useful to analyze the impact of retroviral insertion sites on both malignant and benign clonal dominance of individual repopulating HSC.

Key Words

PCR insertional mutagenesis clonality gene transfer retroviral vector RVISs 



This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG SPP1230, to B.F. and C.B.), the European Union (CONSERT-LSHB-CT-2004-005242, to C.B.), and the National Cancer Institute (R01-CA107492-01A2, to C.B.). We thank Nora Zingler (Heinrich-Pette-Institute, Hamburg, Germany) for technical advice.


  1. 1.
    Baum, C., Schambach, A., Bohne, J., and Galla, M. (2006) Retrovirus vectors: toward the plentivirus? Mol. Ther . 13, 1050–1063.CrossRefPubMedGoogle Scholar
  2. 2.
    Li, Z., Dullmann, J., Schiedlmeier, B., Schmidt, M., von Kalle, C., Meyer, J., Forster, M., Stocking, C., Wahlers, A., Frank, O., Ostertag, W., Kuhlcke, K., Eckert, H. G., Fehse, B., and Baum, C. (2002) Murine leukemia induced by retroviral gene marking. Science 296, 497.CrossRefPubMedGoogle Scholar
  3. 3.
    Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M. P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C. S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., Fraser, P., Cohen, J. I., de Saint Basile, G., Alexander, I., Wintergerst, U., Frebourg, T., Aurias, A., Stoppa-Lyonnet, D., Romana S, Radford-Weiss, I., Gross, F., Valensi, F., Delabesse, E., Macintyre, E., Sigaux, F., Soulier, J., Leiva, L. E., Wissler, M., Prinz, C., Rabbitts, T. H., Le, Deist, F., Fischer, A., and Cavazzana-Calvo, M. (2003) LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419.Google Scholar
  4. 4.
    Kustikova, O., Fehse, B., Modlich, U., Yang, M., Düllmann, J., Kamino, K., von Neuhoff, N., Schlegelberger, B., Li, Z., and Baum, C. (2005) Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 308, 1171–1174.CrossRefPubMedGoogle Scholar
  5. 5.
    Seggewiss, R., Pittaluga, S., Adler, R. L., Guenaga, F. J., Ferguson, C., Pilz, I. H., Ryu, B., Sorrentino, B. P., Young, W. S. 3rd, Donahue, R. E., von Kalle, C., Nienhuis, A. W., and Dunbar, C. E. (2006) Acute myeloid leukemia associated with retroviral gene transfer to hematopoietic progenitor cells of a rhesus macaque. Blood 107, 3865–3867.CrossRefPubMedGoogle Scholar
  6. 6.
    Ott, M. G., Schmidt, M., Schwarzwaelder, K., Stein, S., Siler, U., Koehl, U., Glimm, H., Kühlcke, K., Schilz, A., Kunkel, H., Naundorf, S., Brinkmann, A., Deichmann, A., Fischer, M., Ball, C., Pilz, I., Dunbar, C., Du, Y., Jenkins, N. A., Copeland, N. G., Luthi, U., Hassan, M., Thrasher, A. J., Hoelzer, D, von Kalle, C., Seger, R., and Grez, M. (2006) Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409.CrossRefPubMedGoogle Scholar
  7. 7.
    Abkowitz, J. L., Catlin, S. N., McCallie, M. T., and Guttorp, P. (2002) Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood 100, 2665–2667.CrossRefPubMedGoogle Scholar
  8. 8.
    Schmidt, M., Carbonaro, D. A., Speckmann, C., Wissler, M., Bohnsack, J., Elder, M., Aronow, B. J., Nolta, J. A., Kohn, D. B., and von Kalle, C. (2003) Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nat. Med. 9, 463–468.CrossRefPubMedGoogle Scholar
  9. 9.
    Schmidt, M., Hacein-Bey-Abina, S., Wissler, M., Carlier, F., Lim, A., Prinz, C., Glimm, H., Andre-Schmutz, I., Hue, C., Garrigue, A., Le Deist, F., Lagresle, C., Fischer, A., Cavazzana-Calvo, M., and von Kalle, C. (2005) Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial. Blood 105, 2699–2706.CrossRefPubMedGoogle Scholar
  10. 10.
    Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.CrossRefPubMedGoogle Scholar
  11. 11.
    Steigerwald, S. D., Pfeifer, G. P., and Riggs, A. D. (1990) Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks. Nucleic Acids Res. 18, 1435–1439.CrossRefPubMedGoogle Scholar
  12. 12.
    Schmidt, M., Hoffmann, G., Wissler, M., Lemke, N., Mussig, A., Glimm, H., Williams, D. A, Ragg, S., Hesemann, C. U., and von Kalle C. (2001) Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples. Hum. Gene Ther. 12, 743–749.CrossRefPubMedGoogle Scholar
  13. 13.
    Kustikova, O., Geiger, H., Li, Z., Brugman, M. H., Chambers, S. M., Shaw, C. A., Pike-Overzet, K., de Ridder, D., Staal, F. J. T., von Keudell, G., Cornils, K., Nattamai, K., J., Modlich, U., Wagemaker, G., Goodell, M. A., Fehse, B., and Baum, C. (2007) Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways. Blood 109, 1897–1907.CrossRefPubMedGoogle Scholar
  14. 14.
    Li, Z., Fehse, B., Schiedlmeier, B., Düllmann, J., Frank, O., Zander, A. R,, Ostertag, W., and Baum, C. (2002) Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell. Leukemia 16, 1655–1663.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Olga S. Kustikova
    • 1
    • 2
    • 3
  • Christopher Baum
    • 1
    • 3
  • Boris Fehse
    • 4
  1. 1.Department of Experimental HematologyHannover Medical SchoolHannoverGermany
  2. 2.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia
  3. 3.Division of Experimental HematologyCincinnati Children’s Research FoundationCincinnati
  4. 4.Clinic for Stem Cell TransplantationUniversity Medical Center Hamburg-EppendorfHamburgGermany

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