Retroviral-Mediated Transduction and Clonal Integration Analysis of Human Hematopoietic Stem and Pro

This chapter provides information on the methods used to introduce genes into human hematopoietic stem and progenitor cells, using Moloney Murine Leukemia (MoMuLV)-based retroviral vectors. MoMuLV-based vectors have the ability to efficiently transfer genes into mammalian cells, leading to permanent integration of a single copy of the gene of interest into the cellular chromosomes. The technique of single-colony inverse [polymerase chain reaction (PCR) can be used to track individual descendants of MoMuLV-vector-transduced hematopoietic stem cells (HSC), by capitalizing upon the unique restriction patterns generated by the random integration events (1 –2 ). Methods to adapt the inverse PCR technology to the use of other vector systems, such as lentiviral or adeno-associated virus (AAV) vectors, are currently under development. These techniques will be necessary to determine the efficacy of the newer vector systems in transducing individual human HSC that have the capacity to generate both lymphoid and myeloid progeny, as has been demonstrated in rare occurrences using MoMuLV-based vectors (2 ).