GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells.

Friend virus induces the development of erythroleukemia in mice through the interaction of a viral glycoprotein, gp55, with a truncated form of the Stk receptor tyrosine kinase, short form-Stk (Sf-Stk), and the EpoR. We have shown previously that the ability of Sf-Stk to participate in the transformation of Friend virus-infected cells requires the kinase activity and Grb2-binding site of Sf-Stk. Here we show that Grb2 heterozygous mice exhibit decreased susceptibility to Friend erythroleukemia and that expansion of erythroid progenitors in response to infection requires the C-terminal SH3 domain of Grb2. A fusion protein in which the Grb2-binding site in Sf-Stk is replaced by Gab2, supports the growth of progenitors from mice lacking Sf-Stk, whereas a Sf-Stk/Gab1 fusion protein does not. Gab2 is expressed in spleens from Friend virus-infected mice, co-immunoprecipitates with Sf-Stk and is tyrosine phosphorylated in the presence of Sf-Stk. Mice with a targeted deletion in Gab2 are less susceptible to Friend erythroleukemia and the expansion of erythroid progenitor cells in response to infection can be rescued by expression of Gab2, but not Gab1. Taken together, these data indicate that a Sf-Stk/Grb2/Gab2 complex mediates the growth of primary erythroid progenitor cells in response to Friend virus.

Conjugated linoleic acid decreases production of pro-inflammatory products in macrophages: evidence for a PPAR gamma-dependent mechanism.

Conjugated linoleic acid (CLA) is a dietary fatty acid that has received considerable attention due to its unique properties in rodent models including anti-cancer, anti-atherogenic and anti-diabetic effects. The effects of CLA are similar to those seen with ligands for peroxisome proliferator-activated receptor (PPARs), most notably of the PPAR gamma subtype. With the recent observation of a role for PPAR gamma in regulation of immune responses, we suspected that CLA could affect immune function, in particular macrophage activity. The goal of our study was to examine whether this dietary fatty acid has anti-inflammatory properties similar to those reported for PPAR gamma activators such as 15-deoxy prostaglandin J(2) (PGJ(2)). In reporter assays, various CLA isomers activated PPAR gamma in RAW264.7 mouse macrophage (RAW) cells. CLA decreased the interferon-gamma (IFN gamma)-induced mRNA expression of mediators of inflammation including cyclooxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis factor alpha (TNFalpha). Reporter assays also demonstrated reduced IFN gamma-stimulated transcriptional activity of the iNOS and COX2 promoters by CLA. Consequently, CLA decreased the production of PGE(2), TNFalpha and the inflammatory agent nitric oxide (NO) in RAW cells treated with IFN gamma. Other pro-inflammatory cytokines such as IL-1 beta and IL-6 were similarly decreased by CLA treatment of RAW cells. In addition, various CLA isomers induced HL60 cell differentiation along the monocytic lineage as assessed by measuring expression of the cell surface marker CD14. This differentiation process, as well as the regulation of iNOS and COX2 by 15dPGJ(2), is believed to involve PPAR gamma. Mutations of Leu(468) and Glu(471) to alanine in helix 12 of the ligand-binding domain of PPAR gamma resulted in a protein with strong dominant-negative activity (dnPPAR gamma). Transfecting dnPPAR gamma into RAW cells eliminated the ability of various CLA isomers to regulate the iNOS reporter construct. Taken together, these results suggest that CLA has anti-inflammatory properties that are mediated, at least in part, by the nuclear hormone receptor PPAR gamma.

Muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase in human skeletal muscle.

Physiological stress associated with muscle damage results in systemic insulin resistance. However, the mechanisms responsible for the insulin resistance are not known; therefore, the present study was conducted to elucidate the molecular mechanisms associated with insulin resistance after muscle damage. Muscle biopsies were obtained before (base) and at 1 h during a hyperinsulinemic-euglycemic clamp (40 mU x kg(-1) x min(-1)) in eight young (age 24+/-1 yr) healthy sedentary (maximal O(2) consumption, 49.7+/-2.4 ml x kg(-1) x min(-1)) males before and 24 h after eccentric exercise (ECC)-induced muscle damage. To determine the role of cytokines in ECC-induced insulin resistance, venous blood samples were obtained before (control) and 24 h after ECC to evaluate ex vivo endotoxin-induced mononuclear cell secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Glucose disposal was 19% lower after ECC (P<0.05). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was 45% lower after ECC (P<0.05). Insulin-stimulated phosphatidylinositol (PI) 3-kinase, Akt (protein kinase B) serine phosphorylation, and Akt activity were reduced 34, 65, and 20%, respectively, after ECC (P < 0.05). TNF-alpha, but not IL-6 or IL-1beta production, increased 2.4-fold 24 h after ECC (P<0.05). TNF-alpha production was positively correlated with reduced insulin action on PI 3-kinase (r = 0.77, P = 0.04). In summary, the physiological stress associated with muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase, presumably leading to decreased insulin-mediated glucose uptake. Although more research is needed on the potential role for TNF-alpha inhibition of insulin action, elevated TNF-alpha production after muscle damage may impair insulin signal transduction.

Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells.

Using an expression gene trapping strategy, we have identified and characterized two novel hematopoietic genes, Hzf and Hhl. Embryonic stem (ES) cells containing a gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to generate mice containing the gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C(2)H(2)-type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression trapping to identify novel hematopoietic genes. Future studies of Hzf and Hhl should provide valuable information on the role these genes play during megakaryocytopoiesis.

Negative regulation of macrophage activation in response to IFN-gamma and lipopolysaccharide by the STK/RON receptor tyrosine kinase.

IFN-gamma primes macrophages for antimicrobial activity, increased killing of intracellular pathogens, and Ag processing and presentation to lymphocytes by cooperating with a second signal (provided by LPS or endogenous TNF-alpha) to promote increased proinflammatory cytokine production, NO production, and MHC class II expression. Macrophage-stimulating protein (MSP) suppresses NO production by activated peritoneal macrophages in vitro. Furthermore, targeted deletion of the receptor for MSP, stem cell-derived tyrosine kinase receptor (STK/RON), resulted in increased production of NO by activated macrophages both in vitro and in vivo. Here we demonstrate that expression of STK in RAW264.7 cells resulted in suppression of NO production following IFN-gamma+/- LPS stimulation in the presence of MSP, reflecting a decrease in the levels of inducible NO synthase (iNOS) mRNA and protein, which was confirmed by decreased trans-activation of an iNOS reporter. The iNOS expression is regulated by the coordinate activity of the inducible transcription factors STAT-1, IFN response factor-1, and NF-kappaB. The presence of the STK receptor did not significantly alter the expression of the IFN-gamma receptor, STAT1 phosphorylation, or the up-regulation of IFN response factor-1 expression following IFN-gamma stimulation. However, nuclear translocation of NF-kappaB following stimulation of RAW cells with IFN-gamma and LPS was reduced in the presence of the MSP/STK signaling pathway. These results suggest that the negative regulation of macrophage responses by MSP/STK occurs at least in part via inhibition of costimulatory signals, resulting in NF-kappaB activation, that cooperate with IFN-gamma to promote activation.

Fv2 encodes a truncated form of the Stk receptor tyrosine kinase.

The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.

Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase.

Immune and inflammatory responses must be rightly regulated to maintain a homoeostatic balance between an effective immune response and tissue damage to the host. NO is a principal mediator of many of the cytokine-inducible macrophage activities during a normal cell-mediated immune response. STK, the murine homologue of the human RON receptor tyrosine kinase, is expressed on murine resident peritoneal macrophages. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein that is activated by members of the coagulation cascade in response to tissue damage. In addition to its potential to induce chemotaxis and phagocytosis of C3bi-coated erythrocytes, MSP has an inhibitory effect on the production of NO by activated peritoneal macrophages in vitro. Here we demonstrate that peritoneal macrophages from mice lacking STK produce elevated levels of NO in response to interferon (IFN)-gamma in a dose-dependent manner, without the need for a co-stimulus. However, production of pro-inflammatory cytokines by activated macrophages from stk -/- mice is unaltered. In vivo, stk -/- mice exhibit increased inflammation in an IFN-gamma-mediated delayed-type hypersensitivity reaction and increased susceptibility to lipopolysaccharide (LPS)-induced endotoxic shock. Furthermore, the levels of NO in the serum of mice injected with LPS are significantly higher than those in control littermates. Nevertheless, the serum levels of IFN-gamma and the intermediate cytokines generated by the inflammatory response, which have previously been shown to play a role in septicaemic shock, do not differ significantly from controls. These data suggest that the STK receptor suppresses NO production, therefore ameliorating the potentially tissue-damaging effects of a cell-mediated immune response, through negative regulation of the IFN-gamma signalling pathway.

Retroviral vector design for long-term expression in murine hematopoietic cells in vivo.

A series of retroviral vectors containing the human glucocerebrosidase (GC) cDNA driven by various promoters have been constructed in an attempt to discover which vector design can most efficiently transduce murine hematopoietic stem cells (HSCs) and drive expression of the transferred gene in hematopoietic cells of mice reconstituted with the transduced stem cells. The simplest vector, LG, in which the GC gene is driven by the viral LTR, was the most efficient vector at infecting HSCs, with an average viral copy number in hematopoietic tissues of 3 copies/cell in recipient mice. In general, the viral vectors that contained any additional promoters or enhancers to drive expression of either the GC gene or a selectable marker gene (Neo) had lower titers and/or transduced HSCs at a lower efficiency. This was seen most markedly when the human phosphoglycerate (PGK) promoter was used to drive the human GC cDNA. Despite repeated attempts to obtain a high titer producer clone, this virus consistently produced low titers and subsequently resulted in the lowest proviral copy numbers in long-term reconstituted mice. Only the viral LTR and PGK promoter were capable of driving significant levels of human GC RNA in hematopoietic cells of long-term reconstituted mice, with a much lower level of RNA generated by an internal herpes TK or SV40 immediate early promoter. Insertion of the internal transcription unit in the opposite orientation relative to the viral LTRs had a detrimental effect on gene expression. The levels of RNA generated by a hybrid LTR containing the myeloproliferative sarcoma virus enhancer were higher in bone marrow-derived macrophages than in nonadherent cells of the bone marrow when compared with the LG vector. The presence of an internal promoter to drive expression of the human GC cDNA did not seem to have a detrimental effect on expression levels from the viral LTR. In fact, in the presence of an internal TK or PGK promoter expression from the LTR was increased despite the presence of lower proviral copy numbers. Insertion of a second gene (Neo) into the vector had a negative impact on long-term expression in hematopoietic cells in vivo; however, this seems to be due solely to the lower transduction efficiency of this vector. Overall, the highest levels of GC activity in macrophages of long-term reconstituted mice were generated by the LG vector; however, these levels were variable.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of human glucocerebrosidase in murine macrophages: identification of efficient retroviral vectors.

Gaucher's disease is an autosomal recessive disorder characterized by a functional deficiency in beta-glucocerebrosidase enzymatic activity and the resultant accumulation of the glycolipid glucocerebroside in macrophages. Due to the nature of the affected cells, Gaucher's disease is an excellent candidate for gene therapy of hematopoietic stem cells and autologous bone marrow transplantation of transduced cells using retroviral vectors containing the glucocerebrosidase (GC) gene. In order to identify a retroviral vector capable of high levels of expression of the GC gene in macrophages, we have used the murine myeloid leukemia cell line, M1, a cell line that can be differentiated with interleukin-6 (IL-6) from blasts to macrophages. Two vectors use the Moloney murine leukemia virus (MoMLV) enhancer/promoter (LG vector) or the myeloproliferative sarcoma virus (MPSV) enhancer/MoMLV promoter (MG vector), both located in the viral long-terminal repeat (LTR); the third vector uses the phosphoglycerate kinase (PGK) promoter located internally in the vector (PG vector). The amphotropic PA317 and GP+am12 packaging cell lines were used as virus producer cells, and the GP+am12 cell line demonstrated higher titers, higher levels of GC protein expression, and specific GC enzymatic activity as well as higher transduction efficiencies for all three vectors. The LG retroviral vector was the most efficient in transducing the M1 cells. On average, higher levels of RNA and protein expression were seen in the M1 clones transduced with the LG vector, and these levels increased after differentiation. Thus, the LG retroviral vector in which the expression of the GC gene is driven by the MoMLV LTR enhancer/promoter is the best vector of the three studied for future studies for gene therapy of Gaucher's disease and other hematopoietic disorders that involve macrophages.

Correction of the enzyme deficiency in hematopoietic cells of Gaucher patients using a clinically acceptable retroviral supernatant transduction protocol.

Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase (GC), and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable protocol for this purpose, we created two amplified (A) high-titer retroviral vector-producer cell lines to efficiently transduce hematopoietic stem and progenitor cells. GP+envAm12/A-LGSN (A-LGSN), contained the GC cDNA driven by the retroviral long terminal repeat (LTR) and the neomycin phosphotransferase gene expressed from the simian virus 40 early promoter. GP+envAm12/A-LG4 (A-LG4) contained only the GC gene driven by the LTR. Both A-LGSN and A-LG4 contained multiple proviral copies and gave approximately 10-fold higher titers on 3T3 cells compared to their unamplified counterparts. These vectors were packaged in GP+envAm12 cells because vectors produced in this cell line transduced hematopoietic cells more efficiently than other packaging cells tested. Bone marrow mononuclear cells and purified CD34+ cells were infected with virus supernatants four times in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) over 96 hours in culture. Cells were then plated in semisolid cultures and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies were scored for vector presence by polymerase chain reaction (PCR). Transduction efficiency of CFU-GM colonies derived from CD34+ cells was improved considerably using the amplified vectors in the GP+envAm12 packaging line. For A-LGSN, A-LG4, and unamplified LGSN, transduction efficiencies were 41, 42, and 25%, respectively. Therefore, multiple proviral copies resulting in higher titer improves retroviral transduction of human hematopoietic progenitor cells. Hematopoietic cells from Gaucher patients were transduced and placed into long-term bone marrow culture (LTBMC). Viral supernatant from the amplified producer lines transduced long-term culture initiating cells (LTCIC) efficiently (30 to 50%) using this clinically acceptable protocol. Both sustained mRNA expression and GC enzyme production are achieved in the long-term culture of LTCIC and lead to correction of the GC deficiency in their progeny cells.

Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages.

The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.

Gene transfer and bone marrow transplantation with special reference to Gaucher's disease.

Gene therapy following gene transfer into hematopoietic cells is now being investigated for several genetic disorders (1). The candidate diseases under consideration and active experimental investigation are disorders that can be fully or partially corrected by allogeneic bone marrow transplantation, where the gene defect is known and the normal gene has been cloned and characterized. Together with ADA deficiency Gaucher's disease is a leading candidate among these disorders. We have developed high titer retroviral vectors containing the glucocerebrosidase gene that can transduce murine bone marrow stem cells with high efficiency and will result in high levels of human glucocerebrosidase in macrophages of long-term reconstituted mice. "Gene therapy" for Gaucher's disease has therefore been accomplished in the mouse. We have also developed high titer amphotropic vectors containing the glucocerebrosidase gene that can transduce human CD34+ hematopoietic progenitors with a high degree of efficiency. Up to 70% of CFU-GM colonies from these infected CD34+ cells contained the transferred gene as determined by PCR. Our vectors can also correct the enzyme deficiency of hematopoietic cells from Gaucher patients following gene transfer.

High levels of human glucocerebrosidase activity in macrophages of long-term reconstituted mice after retroviral infection of hematopoietic stem cells.

Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit-spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttransplantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respectively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the human GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of directing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggesting that this virus may be useful in the treatment of Gaucher disease.

Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease.

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py+/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hematopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py+/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection (by means of the polymerase chain reaction) of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py+/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity had been normalized.

Expression of human glucocerebrosidase in long-term reconstituted mice following retroviral-mediated gene transfer into hematopoietic stem cells.

A retroviral vector (GTN) in which the glucocerebrosidase (GCase) cDNA is driven by the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) was tested for transfer efficiency and expression of the GCase gene in long-term reconstituted mice. Eleven W/Wv mice were transplanted with unselected GTN-infected bone marrow cells and 10 of these mice were analyzed 3 months later. Seven of these 10 mice (70%) contained the intact proviral genome in bone marrow, spleen, and thymus. Of these 7,3 mice contained a high-copy number of the provirus in all the hematopoietic tissues tested. The mice contained anywhere from one to four proviral integration sites that were the same in all three tissues, indicating that these mice have been repopulated by one or more transduced multipotential hematopoietic stem cells. Five months after transplantation, bone marrow from the eleventh mouse was transplanted into secondary recipient animals. The secondary recipients contained the intact proviral genome in the bone marrow, spleen, thymus, and macrophages 4 months after the secondary transplantation. This further supports the conclusion that hematopoietic stem cells have indeed been targeted. Human GCase RNA was detected in all 7 mice containing the proviral DNA. These results demonstrate expression of the human GCase gene in the progeny of repopulating hematopoietic stem cells of mice following gene transfer.

Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells.

The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Mouse bone marrow cells were stimulated with hematopoietic growth factors, infected by coculture with producer cells, and injected into lethally irradiated animals. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to stimulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. When an internal herpes simplex thymidine kinase promoter assisted by a mutant polyoma enhancer was used to drive the human GC cDNA (NTG vector), little or no human GC RNA was detected in transduced CFU-S colonies. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. NTG vector-infected bone marrow cells were transplanted into W/Wv recipients to generate long-term reconstituted mice. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.