Retroviral Integration Site Analysis in Hematopoietic Stem Cells

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.