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The mouse is the most commonly used experimental animal, and a wide range of tumor types can arise in their hematopoietic system. Therefore, for research scientists and graduate students working in the field of experimental hematology, immunology, and cancer research, there is an urgent need for well-established protocols for the preparation of histology and cytology for leukemia diagnosis. Moreover, the criteria for the classification of hematopoietic neoplasms often vary between different laboratories. In this chapter, we describe diagnosis and analysis of leukemia in murine bone marrow transplantation models based primarily on the findings of the histology and cytology of hematopoietic and infiltrated tissues, peripheral blood smear, and immunophenotyping by FACS analysis.

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation and gene therapy applications. Their progeny cells carry the genetic information of the HSCs and replenish the blood and immune system. Therefore, in the setting of inherited diseases, transduction of HSCs with retroviral vectors (including gammaretro- and lentiviral vectors) offers the possibility to correct the phenotype in all blood lineages as demonstrated in clinical trials for immunodeficiencies (e.g., X-SCID). In the process of developing gene therapy strategies for patient applications, suitable mouse models for the human gene therapy are important to validate the concept. Stem-cell-enriched populations such as lineage negative cells as the functional equivalent of human CD34(+) cells can be isolated from murine bone marrow and efficiently transduced using retroviral vectors. This chapter provides a step-by-step protocol for retroviral transduction of murine lineage negative cells.

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.