Summary
Identification of retroviral vector insertion sites in single, dominating cell clones has become an important tool for the investigation of cellular signalling pathways involved in clonal expansion and malignant transformation. Also, recent severe adverse events in clinical trials resulting from retroviral vector-mediated insertional mutagenesis underline the need of well-designed safety studies including integration site analyses to estimate cost/benefit ratios in gene therapy. We have recently described a modified ligation-mediated PCR (LM PCR) method allowing preferential retrieval of insertion sites causally linked to clonal dominance of an affected clone. In the first part of the given work we focus on particularities of the LM PCR procedure to be taken into account when working with self-inactivating as compared to ‘classical’ retrovectors. In the following sections we focus on data acquisition, processing, organisation, and analysis. Thus the protocol presented here should be helpful in establishing and utilising databases of retroviral integration sites.
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Acknowledgments
This work was supported by Grants from the Deutsche Forsc-hungsgemeinschaft (DFG SPP1230). The authors wish to thank Martijn Brugman and Hartmut Geiger for critical reading of the manuscript.
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Kustikova, O.S., Modlich, U., Fehse, B. (2009). Retroviral Insertion Site Analysis in Dominant Haematopoietic Clones. In: Baum, C. (eds) Genetic Modification of Hematopoietic Stem Cells. Methods In Molecular Biology™, vol 506. Humana Press. https://doi.org/10.1007/978-1-59745-409-4_25
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DOI: https://doi.org/10.1007/978-1-59745-409-4_25
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Print ISBN: 978-1-58829-980-2
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