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        Genetic Modification of Murine Hematopoietic Stem Cells by Retroviruses

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        Among the currently available methods for gene transfer, recombinant murine retroviruses remain the best established method for achieving stable integration of a transgene with high efficiency. Pioneering work by a number of groups has demonstrated the feasibility of using this method for gene transfer to primitive, multipotential long-term repopulating hematopoietic stem cells (HSC) (1 4 ). In the case of the hematopoietic system, it is required that the introduced gene integrates into the genome of HSC in order to be expressed in multiple lineages over an extended period of time. However, HSC are found at low frequency, and are normally in a quiescent or slow cycling state. Both factors represent challenges to successful retroviral gene transfer. The former places a premium on high titer, and the latter dictates methods to trigger HSC cycling during the infection, since stable integration of murine retroviruses requires cell division of the target cell and breakdown of the nuclear membrane (5 ,6 ). In general, titers greater than 1 � 105 U/mL allow some degree of gene transfer for HSC, but 1 � 106 or higher are a reasonable goal for achieving useful efficiencies of at least 20%. For activation of HSC, most protocols invoke a combination of in vivo and in vitro stimulation. The former is most easily and routinely achieved by administration of cytotoxic agents like 5-fluorouracil (5-FU) 4 d prior to bone-marrow harvest. This procedure removes a large proportion of actively cycling, more differentiated cells, thus achieving a
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