Retroviral insertion site analysis in dominant haematopoietic clones.
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Retroviral insertion site analysis in dominant haematopoietic clones. / Kustikova, Olga S; Modlich, Ute; Fehse, Boris.
In: Methods Mol Biol, Vol. 506, 2009, p. 373-390.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Retroviral insertion site analysis in dominant haematopoietic clones.
AU - Kustikova, Olga S
AU - Modlich, Ute
AU - Fehse, Boris
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
M3 - SCORING: Zeitschriftenaufsatz
VL - 506
SP - 373
EP - 390
JO - Methods Mol Biol
JF - Methods Mol Biol
SN - 1064-3745
ER -