Therefore, these experiments did not provide any evidence for the BAG infection of myeloid or multipotential hematopoietic progenitors. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 MSI-1436 lactate or CD34, as well as in osteoclasts. Moloney murine leukemia virus (M-MuLV) is a simple retrovirus that induces T lymphoma in susceptible mice. Leukemogenesis by M-MuLV has been studied extensively (reviewed in reference 6). It has become clear that it is a multistep process, with several well-defined events taking place in an orderly fashion. Two well-recognized events include the insertional activation of proto-oncogenes and the generation of polytropic envelope recombinants (MCF recombinants [7]) in the infected animal. We have employed an MSI-1436 lactate enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, to study M-MuLV leukemogenesis in mice. This virus contains enhancer sequences from the F101 strain of murine polyoma virus inserted into the M-MuLV long terminal repeat downstream of the M-MuLV enhancers (11). Mo+PyF101 M-MuLV shows substantially attenuated leukemogenicity when inoculated subcutaneously (s.c.) into newborn mice (3, 5). Comparative studies with Mo+PyF101 and wild-type M-MuLV have identified a series of preleukemic events induced by wild-type M-MuLV, notably hematopoietic hyperplasia in the spleen. The splenic hyperplasia appears to result secondarily from stromal defects in the bone marrow (10). The leukemogenic defect of Mo+PyF101 M-MuLV is also dependent on the route of inoculation. When Mo+PyF101 M-MuLV is inoculated s.c. it shows attenuation; when inoculated intraperitoneally (i.p.) it shows leukemogenicity equivalent to that of wild-type M-MuLV (1). Comparative studies of mice inoculated s.c. and i.p. with Mo+PyF101 M-MuLV provided further insight (1). The rate of infection for Mo+PyF101 M-MuLV in the thymus (the ultimate target MSI-1436 lactate organ for M-MuLV leukemogenesis) did not differ between s.c. and i.p. inoculation. On the other hand, early infection (1 to 2 2 weeks) in the bone marrow was substantially reduced in mice infected s.c. with Mo+PyF101 M-MuLV compared to those infected by the i.p. route. This indicated that early bone marrow infection is essential for efficient leukemogenesis by M-MuLV. One possibility is that the bone marrow might seed infection to lymphoid precursors that subsequently migrate to the thymus. In light of the identification of the bone marrow as a critical target for M-MuLV infection, we were interested in a more detailed characterization of early bone marrow infection. In particular, we were interested in identifying the cell types that become infected and the order in which this occurs. To identify the first cells infected, we employed a replication-defective M-MuLV-based retroviral vector (BAG) that expresses a readily detectable reporter gene, the gene that encodes bacterial -galactosidase. In vivo infection with this vector allows the identification of cells that are directly infected by the injected virus, since the vector cannot spread to other cells. Infection of the bone marrow after i.p. inoculation with an M-MuLV-based retroviral vector is characterized in this report. MATERIALS AND METHODS Viruses and inoculation of mice. Psi-2 cells (12) were transfected EPHB4 by a plasmid containing the BAG vector (kindly provided by Constance Cepko [15]) and were selected for the presence of the vector by growth in a medium containing G418. G418-resistant cells were grown in Dulbecco modified Eagles medium (DMEM) supplemented with 10% calf serum as described previously (15). The cell culture supernatant was harvested and concentrated 10- to 20-fold by ultrafiltration with an Amicon Centriprep 50 (Amicon Inc., Beverly, Mass.). To titrate viral supernatants, MSI-1436 lactate serial dilutions were used to infect NIH/3T3 cells that had been pretreated for 1 h with 20 g of Polybrene per ml. Cells were allowed to grow until confluence and were stained for -galactosidase activity as described below, and the number of blue colonies was counted. Viral vector titers of 2 106 to 6 106 infectious units/ml were routinely obtained. These viral supernatants were used to inoculate neonatal NIH/Swiss mice i.p. (200 l per animal). Mice were sacrificed at various.