Abrogation of the requirement for feeder cell interaction and T cell receptor stimulation of lymphocytes infected with retroviral vectors.

Infection of sensitive adult mice with myeloproliferative sarcoma virus (MPSV) results in a myeloproliferative syndrome. Two components of the viral genome are required to induce this unique pathology: the mos oncogene and sequences within the U3 region of the long terminal repeat (LTR). In studies designed to identify the target cell of MPSV and thus better understand the mechanism by which a myeloproliferative syndrome is induced, we have infected a series of T cell lines with MPSV-based vectors. The results presented here show that infection with neoR MPSV abrogates the requirement for an antigen-specific or feeder cell-dependent stimulation, without altering the requirement for interleukin 2. Significantly, this response is not dependent on the mos oncogene, but requires sequences within the U3 region of the MPSV LTR. No alteration in the constitutive or induced levels of lymphokines released by these cells was observed. These results suggest a model in which T cells acquire a proliferative advantage by uncoupling the proliferative response from the lymphokine synthesis that is induced by activation of the T cell receptor. These cells are thus poised for antigen stimulation and secretion of cytokines that stimulate myelopoiesis.

Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene.

A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Lack of retrovirus gene expression in somatic cell hybrids of friend cells and teratocarcinoma cells with a teratocarcinoma phenotype.

Two types of hybrids between cells with erythroid phenotype (Friend cells) and teratocarcinoma cells can be distinguished: cell hybrids with an erythroid phenotype, which release or can be induced to release large amounts of Friend spleen focus-forming virus (F-SFFV) on exposure to bromodeoxyuridine and cell hybrids with a teratocarcinoma phenotype, which do not release Friend virus and are not inducible for F-SFFV release. In this paper, we attempted to relate these differences to the expression of F-SFFV and Friend murine leukemia virus (F-MuLV) functions. Teratocarcinoma phenotype hybrids retained F-SFFV-and F-MuLV-related provirus sequences. They did not express F-SFFV- or F-MuLV-related RNA or proteins. The hybrids differentiated to endoderm-like cells on exposure to retinoic acid or hexamethylene-bis -acetamide. These cells, in contrast to the teratocarcinoma phenotype (uninduced) cells expressing SSEA-1-like antigens, did not express SSEA-1-like antigens; they formed typical, prekeratin-staining cytoskeletal structures and could be induced to release mouse interferon. The differentiating cells, but not the uninduced teratocarcinoma hybrids, were infected productively with F-MuLV or the F-MuLV--F-SFFV complex. They, however, did not express endogenous F-SFFV. Endogenous F-SFFV functions could not be rescued by infection with F-MuLV. Induction of teratocarcinoma hybrids with retinoic acid did not activate endogenous F-MuLV or F-SFFV transcription or protein synthesis. These data demonstrated two control mechanisms of Friend virus repression: one which acted trans during formation of the cell hybrids and was maintained only in teratocarcinoma phenotype cells and the other which acted cis and was still operative during induction of endodermal differentiation.

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.

Friend erythroleukemia virus complex: role of viral components in modifying erythroid differentiation in mice.

The present study was carried out to determine which component of the polycythemic strain of Friend erythroleukemia virus (FV-P)--Friend helper virus [Friend murine leukemia virus (F-MuLV)] of the replication-defective spleen focus-forming virus (SFFV)--is responsible for inducing erythropoietin-independent erythropoiesis and expansion of the erythroid compartment in infected mice. F-MuLV and SFFV were cloned in SC1 cells to give a cell line 643/22N, which released only F-MuLV and SC204, a nonproducer cell line carrying SFFV. On superinfection with specific helper viruses, SC204 yielded further cell lines, which released SFFV in conjunction with 1) Friend, 2) Moloney, or 3) Gross helper viruses. With the use of an in vitro colony formation assay to monitor erythropoiesis in the bone marrow of inbred DBA/2J mice infected with any of the virus preparations containing SFFV plus helper virus, the erythroid precursor cell population was found to become erythropoietin-independent and amplified; adult mice infected with any of the helper viruses alone did not develop any symptoms of Friend disease. Thus the biologic activity of the FV-P complex was unaffected by replacement of the F-MuLV with an unrelated helper virus (Moloney or Gross). These results indicated that the SFFV component of FV-P is responsible for modifying erythroid differentiation in adult mice.

Effects of myeloproliferative sarcoma virus on the pluripotential stem cell and granulocyte precursor cell populations of DBA/2 mice.

The hematopoietic stem cell (CFU-S) and granulocyte precursor cell (CFU-C) populations have been assayed in the spleen, blood, and bone marrow of DBA/2 mice at various times after infection with the myeloproliferative sarcoma virus (MPSV). Beginning between 7 and 19 days after virus infection, the number of CFU-S showed a steady, parallel increase in the blood and spleen, reaching a maximum at both sites by days 25-30. At the maximum, in the spleen the concentration of CFU-S was 10 times greater than that in the blood, and the total number of CFU-S was over 100 times greater than that of normal animals. During the same period, in the bone marrow the number of CFU-S decreased to one-half of normal. Nevertheless, the CFU-S from MPSV-infected animals differentiated normally in the spleens of irradiated, normal recipient mice (except for some hyperplasia of the erythroid component of spleen colonies). The CFU-C content of the bone marrow, spleen, and blood paralleled the CFU-S content of these organs: The CFU-S and CFU-C populations changed almost synchronously after MPSV infection. In the terminal stage of the MPSV-induced disease, a variable proportion of the CFU-C population acquired the ability to differentiate in the absence of added colony-stimulating factor.

Particle-associated RNA dependent DNA polymerase and high-molecular-weight RNA in a human cell line derived from polycythemia vera bone marrow.

A particle fraction with a density of 1.15-1.19 g/cm3 was isolated from the cytoplasm of a human cell line established in culture from the bone marrow of an untreated patient with polycythemia vera. Electron micrographs of cross sections of cells and cell homogenates revealed virus-like particles on which DNA could be synthesized. An RNA-dependent DNA polymerase, isolated from the particles, preferred poly(rA)-oligo(dT) over poly(dA)-oligo(dT) and was able to polymerize deoxyguanosine monophosphate in a reaction stimulated by poly(rC)-oligo(dG).

Transcriptional regulation of the c-fms proto-oncogene mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) in murine cell lines.

Differentiation of blood cells is paralleled by a timely ordered expression of cytokine receptor genes. We show here that the expression of the c-fms gene which encodes the lineage-specific receptor for macrophage colony-stimulating factor (M-CSF or CSF-1) is directly linked to ligand-mediated activation of the receptor for the granulocyte/macrophage colony-stimulating factor (GM-CSF). In interleukin-3 (IL-3) dependent multipotent progenitor cells, FDC-Pmix GMV#2 cells, GM-CSF treatment results in the rapid formation of full-length c-fms transcripts. Surprisingly, this upregulation of c-fms transcripts is also observed in mouse NIH3T3 fibroblasts stably transfected with genes coding for the alpha- and beta-subunits of the GM-CSF receptor. These results indicate a direct control by the GM-CSF receptor that takes place regardless of cell differentiation. Furthermore, a 2.1 kb genomic fragment containing the c-fms proximal promoter directs GM-CSF-inducible expression of a reporter gene, suggesting a regulation of c-fms gene expression on the transcriptional level.

Retrotransposons as mutagens in the induction of growth autonomy in hematopoietic cells.

Factor-independent mutants of hematopoietic cells, especially of multipotent cells, are valuable tools to identify genes that regulate stem cell proliferation and differentiation and thus may be important in leukemogenesis. Factor-independent mutants from both myeloid precursor and hematopoietic stem cell lines were isolated. The frequency of such mutants in a given cell population was one to two orders of magnitude lower for the multipotent cell line FDC-Pmix (3.6 x 10(-9)) than for the myeloid precursors, FDC-P1-M (1.7 x 10(-8)) and D35 (2.2 x 10(-7)). Analysis of these mutants revealed several mechanisms by which growth autonomy was obtained, either with or without direct contribution of growth factor gene activation. The molecular basis of spontaneous activation of the Multi-CSF (Interleukin3) gene was determined and compared to activation of the GM-CSF gene in a previous study. Multi-CSF gene activation in both precursor and stem cells was caused by the insertion of an intracisternal A particle (IAP) provirus. In two independent mutants of the D35 cell line, activation of the Multi-CSF or the GM-CSF gene was caused by almost identical IAPs with a 99% homology in the U3 and R region of the long terminal repeat. This result demonstrates that only one class of IAPs, or perhaps a single provirus, is involved in transposition and gene activation in a particular cell line. A unique example of anti-sense promotion from an IAP provirus in one Multi-CSF mutant underlines the versatility of these elements as natural insertional mutagens.

Conversion of factor-dependent myeloid cells to factor independence: autocrine stimulation is not coincident with tumorigenicity.

It has been postulated that the disruption of the normal hormonal regulation of blood cell formation and proliferation leads to the autonomous growth of hematopoietic progenitors or stem cells and thus to leukeamia. We have utilized established hematopoietic cell lines to establish the different mechanism by which growth autonomy is acquired. The analysis of thirteen spontaneous factor-independent mutants revealed that the majority (12/13) secreted a factor that stimulated growth of the parental cell line. Thus, autocrine stimulation may be a important mechanism by which normal growth control is disrupted. This is supported by the observation of Young and Griffin (1987) that some cells isolated from patients with acute myeloblastic leukemia (AML) autogenously produce growth factor. In the majority of Dind mutants more closely examined, growth factor gene activation was due to the juxtapostion of a retrotransposon. Although the exact nature of the involvement of human retroviruses in inducing leukemia has not been elucidated, one could envisage that altered growth factor regulation due to integration of the virus may play an important role. The existence of a second class of Dind mutants that have obtained factor-independence by a mechanism not involving factor production concurs with the acquisition of factor-independent growth in hematopoietic cells after introduction of some oncogenes. Several models have been proposed to explain how oncogenes may "short circuit" and thus activate the normal signal transduction pathway by mimicking the active receptor, transducer, or effector (Weinberg, 1985). To investigate more closely the role of autocrine stimulation in the induction of growth autonomy and tumorigenicity, retroviral vectors expressing either GM-CSF or IL3 were introduced into factor-dependent hematopoitic cell lines. Non-linear clonability of infected cell lines in the absence of exogenous growth factor and inhibition of proliferation by antiserum supported a model of autocrine stimulation. However, a secondary event, correlated with amount of factor released, often occurred that abrogated the requirement for secreted CSF. Growth of cells in which this alteration had occured was cell-density independent and could not be blocked by antibody. It has been postulated that autogenous factor may react with its receptor intracellularly (Lang et al., 1985). The results presented here cannot exclude that the secondary events may allow the internal interaction of receptor and factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the stem cell factor (SCF) receptor and the alternative forms of its ligand (SCF) in the induction of long-term growth by stroma cells.

Self renewal and differentiation of hematopoietic stem cells is regulated by the hematopoietic microenvironment. The Stem Cell Factor (SCF) has been shown to be one of the essential factors that governs stem cell maintenance. In this abstract we describe a functional analysis of the membrane bound and soluble SCF within the context of stroma cocultures with hematopoietic cells. We report that transmembrane SCF is essential for long term growth, whereas soluble SCF supports only short term proliferation of stroma dependent hematopoietic cells. We also show that the SCF/c-kit interaction can be substituted by an unknown mechanism. To determine the molecular mechanism involved in maintenance of hematopoietic cells on stroma we analyzed the c-kit expression during coculture. We demonstrate that c-kit expression was downregulated during coculture. Downregulation was induced by the coculture itself and not by external factors.

Mechanisms of growth factor expression in acute myeloid leukemia (AML).

To investigate possible mechanisms of growth factor expression in acute myeloid leukemia, genes for granulocyte macrophage colony-stimulating factor (GM-CSF) were analyzed by Southern blots in 20 patients, for M-CSF in 13, for interleukin-6 (IL-6) in 14, for IL-6 receptor in 14 and for G-CSF in five patients. Only in one patient a complex rearrangement of the G-CSF gene with possible amplification was noted indicating rarity of direct alterations of growth factor genes in acute myelogenous leukemia (AML). Spontaneous m-RNA expression for GM-CSF was found in only one of 20 patients, and for IL-6 in eight of 11 patients. In vitro incubation of AML cells of eight patients with recombinant tumor necrosis factor for 24 hr revealed induction of GM-CSF m-RNA expression in three cases and GM-CSF protein expression in two of them. These data suggest that spontaneous GM-CSF production occurs rarely in AML and that monokines, such as tumor necrosis factor, may induce GM-CSF in AML cells. Therefore, interactions of AML cells with normal or malignant accessory cells may be important for autocrine stimulation in AML. Our data suggest that ectopic growth factor secretion is not the primary cause of generating AML but may contribute to progression of the disease. Alternatively, AML may represent a heterogenous group of leukemias with different etiology but similar phenotype.

Murine retrovirus-induced malignant histiocytosis, an experimental model for the disease in humans.

The hematopoietic disregulation in adult mice induced by the malignant histiocytosis sarcoma virus (MHSV) and the Harvey murine sarcoma virus (Ha-MuSV), which both possess c-Ha-ras-related oncogenic sequences, was investigated. Spleen focus formation induced by MHSV and Ha-MuSV was not restricted by the Fv-2 resistance locus in congenic DDD and C57BL/6 mice, unlike leukemogenesis induced by Friend virus, Rauscher virus, and the myeloproliferative sarcoma virus (MPSV). C57BL/6 mice were much more resistant to MHSV and Ha-MuSV-induced spleen focus formation than DDD mice regardless of their Fv-2 state. Infection of DDD mice with MHSV caused a systemic histiocytic neoplasia, best described as murine malignant histiocytosis. Transformed histiocytic cells proliferated excessively in the bone marrow, spleen, and lymph nodes and, in the final stages of the disease, in all major parenchymal organs. The Ha-MuSV caused a strikingly different benign histiocytic tumor in DDD mice and, unlike MHSV, did not induce a rapid, progressive splenomegaly in C57BL/6 mice. Infection of DDD mice with MHSV induced a rapid and synchronized depletion of early and late erythroid precursor cell pools. In MHSV-infected C57BL/6 mice comparable changes were observed with dissimilar kinetics. Macrophage colony-forming cells of MHSV-infected mice were increased in number and proliferated independently of stimulating growth factors. The disease induced by MHSV in mice can thus serve as a model for malignant histiocytosis in humans.

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

The role of soluble growth factors in inducing transient growth and clonal extinction of stroma cell dependent erythroblastic leukemia cells.

A coculture system of a murine erythroblastic leukemia cell line (ELM-D) with its supportive stromal cell line (MS-5) was established. Long-term growth of ELM-D cells is strictly stroma cell dependent. Interaction between stem cell factor (SCF) and its receptor, c-kit, was demonstrated to be important for stroma cell-dependent growth by anti c-kit neutralizing monoclonal antibody (mAb) inhibition experiments. Significantly, soluble growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or SCF of MS-5 stromal cells (MS-5 CM) could replace the requirement of stroma cells for a considerable period. However, ELM-D cells maintained in these growth factors underwent clonal extinction after 3-6 weeks unless contact with stroma was re-established. Furthermore, IL-3 or GM-CSF acted in a dominant manner in inducing cell death in the presence of stroma cells. Cells showing clonal extinction undergo programmed cell death and do not differentiate. These altered growth properties of ELM-D cells exposed to soluble growth factors or to stroma cells appear to be analogous to those described for T or B cells primed by antigen presenting cells and then grown in growth factors.

Virus-transformed macrophage-like cell line as tool for eicosanoid research.

The ability of a virus-transformed murine macrophage like cell line HA 38 to produce different eicosanoid metabolites was examined. HA 38 cells release similar amounts of prostaglandins and leukotrienes as did murine peritoneal macrophages in response to both physiological and non-physiological stimuli. Enzyme systems known to be involved in the regulation of eicosanoid synthesis are expressed. HA 38 cells thus are a well defined macrophage model system and are well suited to study eicosanoid synthesis in macrophages and effects of drugs on the prostaglandin and leukotriene synthesis pathways.

Activity of Friend mink cell focus-forming retrovirus during myelo-erythroid hematopoiesis.

Friend mink cell focus-forming (FMCF) viruses are recombinants between the Friend murine leukemia virus (F-MuLV) and endogenous polytropic retroviruses involved in a number of retrovirus-induced malignancies of the myelo-erythroid compartment. To analyze the contribution of the viral cis regulatory elements to the host range determinants within the hematopoietic system, we performed a series of marker gene experiments using both transient transfection and retroviral-mediated stable transduction of indicator cell lines representing distinct developmental stages. According to our data, the U3 region in the long terminal repeat (LTR) of FMCF viruses possesses an enhancer assembly that allows efficient transcription in both early and late myelo-erythroid stem and progenitor cells. Retroviral gene expression, however, is subjected to stage-dependent transcriptional controls during blood cell maturation. We obtained evidence that a repressor element overlapping with the primer binding site in the viral leader region compromises U3-mediated gene expression in a stage-dependent manner, with the strongest restriction observed in the most primitive cells analyzed, FDCP-mix. In addition, our data indicate a second hurdle for retroviral gene expression in early hematopoietic cells that is independent of the primer binding site and most likely related to inefficient utilization of U3-located enhancers. These data shed light on the mechanisms of host range restriction within the hematopoietic system and define a basis for the design of retroviral vectors aimed to overcome transcriptional inefficiency in early hematopoietic cells. Thus, we developed novel retroviral vectors combining FMCF-type U3 regions with a permissive leader from the murine embryonic stem cell virus. These vectors are highly efficient for gene transfer and expression in both early and late myelo-erythroid cells, indicating that they will be of great use for a variety of experimental and therapeutic applications.