Summary
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.
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References
Baum, C., Schambach, A., Bohne, J., and Galla, M. (2006) Retrovirus vectors: toward the plentivirus? Mol. Ther . 13, 1050–1063.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Acknowledgments
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.
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Kustikova, O.S., Baum, C., Fehse, B. (2008). Retroviral Integration Site Analysis in Hematopoietic Stem Cells. In: Bunting, K.D. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Biology™, vol 430. Humana Press. https://doi.org/10.1007/978-1-59745-182-6_18
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DOI: https://doi.org/10.1007/978-1-59745-182-6_18
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