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X J Yu

Publications and source records attributed to X J Yu.

At least 55 records · Page 3Linked to original sources
PubMed1998

Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2 degreesCFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2 degreesCFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2 degreesCFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2 degreesCFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2 degreesCFU-S from 8 recipients and expression in >50% of the vector-containing 2 degreesCFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.

Animals↗
PubMed1998

Detection of a spotted fever group rickettsia in Amblyomma cajennense (Acari: Ixodidae) in south Texas.

Thai tick typhus rickettsia (strain TT-118), a spotted fever group rickettsia of indeterminant pathogenicity, was isolated from a mixed pool of larval Ixodes and Rhipicephalus ticks collected from Thailand in 1962. Here we report the surprising finding of a spotted fever group rickettsia with closest sequence homology to the Thai tick typhus rickettsia in Amblyomma cajennense (F.) ticks from south Texas. Sequence analysis was performed on segments of 3 genes that differentiate rickettsial species; all 3 genes sequenced, the 17-kDa, glta, and rompA, when compared with those of other rickettsiae, showed the highest degree of similarity to the Thai tick typhus rickettsia with 99.5, 99.5, and 100% homology, respectively. This is the 1st finding of a rickettsial species in A. cajennense ticks in Texas and the United States. If this rickettsia is pathogenic, exposure to infected A. cajennense ticks may pose a previously unrecognized health risk to people who have been fed upon by these ticks.

Animals↗
PubMed1997

Sequence and characterization of an Ehrlichia chaffeensis gene encoding 314 amino acids highly homologous to the NAD A enzyme.

DNA sequence analysis of the nadA gene of Ehrlichia chaffeensis revealed a 942 bp open reading frame with the capacity to encode 314 amino acids. The amino acid sequence of the E. chaffeensis quinolinate synthetase A (NAD/A) has 53.6% identity and 82% similarity to the NAD A of the cyanelle of Cyanophora paradoxa. Portions of the homologous genes of E. canis and E. muris were also sequenced. The amino acid sequences of the NAD A of E. canis and E. muris have 89.2% and 93.2% homology, respectively, to the NAD A of E. chaffeensis. We propose that the nadA gene may be an excellent candidate for a genetic tool for the phylogenetic study of ehrlichiae.

Bacterial Proteins↗
PubMed1997

Cloning and sequencing of the gene for a 120-kDa immunodominant protein of Ehrlichia chaffeensis.

Ehrlichia chaffeensis is the tick-borne, obligately intracellular bacterium that causes human monocytic ehrlichiosis. A 120-kDa protein is one of the immunodominant proteins of E. chaffeensis that stimulates production of specific antibodies in infected humans. A genomic library of E. chaffeensis was constructed in a lambda ZAP II phage vector, and a clone expressing the 120-kDa protein of E. chaffeensis was identified using canine anti-E. chaffeensis serum. DNA sequence analysis of the cloned 120-kDa protein gene of E. chaffeensis identified a 1884-bp open reading frame with an ehrlichial promoter. Five identical 240-bp tandem repeat units were identified in the 120-kDa protein gene of E. chaffeensis, comprising 60% of the entire gene. Aside from the first repeat unit, all the other repeat units are identical. In the first repeat unit there are four nucleotides that are different from the other repeats. Hydropathy analysis of the deduced amino-acid sequence demonstrated that the repeat domain contains highly hydrophilic segments. The 120-kDa protein should be evaluated for a role in stimulating protective immunity.

Amino Acid Sequence↗
PubMed1997

The 5-HT1D receptor antagonist GR-127,935 prevents inhibitory effects of sumatriptan but not CP-122,288 and 5-CT on neurogenic plasma extravasation within guinea pig dura mater.

The aim of this study was to examine whether GR-127,935, a 5-HT1B/1D receptor antagonist, blocks the inhibitory effects of sumatriptan, CP-122,288 and 5-carboxamidotryptamine (5-CT) on plasma protein extravasation, within guinea pig and rat dura matter, following electric stimulation of the trigeminal ganglion. Binding studies first established that GR-127,935 shows a 500-fold selectivity for 5-HT1D binding sites (labeled by [3H]L-694,247) versus 5-HT1F binding sites (labeled by [3H]sumatriptan in the presence of 50 nM 5-carboxamidotryptamine) in guinea pig forebrain homogenates (pKD +/- SD = 7.0 +/- 0.2 at 5-HT1F sites and 9.7 +/- 0.1 at 5-HT1D sites). In guinea pigs, GR-127,935 showed partial agonist activity and inhibited dural plasma protein extravasation. Increasing doses of GR-127,935 reversed the effect of sumatriptan, but did not affect the action of 5-CT and CP-122,288 (at a dose as high as 2 mumol/kg). Sumatriptan, CP 122,288 and 5-CT dose-responsively inhibited plasma protein extravasation. At a dose of 2 mumol/kg (but not at 0.2 mumol/kg), GR-127,935 right-shifted the dose-response curve of sumatriptan. No significant rightward shift was observed in the dose-response of CP-122,288 and 5-CT. In rats, GR-127,935 did not show any significant partial agonist activity. A dose of 0.2 mumol/kg was sufficient to right-shift the dose-response curve of sumatriptan. These data suggest that sumatriptan inhibits neurogenic inflammation via 5-HT1D alpha receptors in guinea pigs and 5-HT1D beta (5-HT1B) receptors in rats. Additional receptor subtypes are likely to be involved in the inhibition of plasma extravasation by CP-122,288 and 5-CT. Pertussis toxin reduced the inhibitory effects of both sumatriptan and 5-CT, but not of muscimol, known to act at GABAA receptors. These results suggest that 5-CT, as well as sumatriptan, act at a receptor linked to an inhibitory G-protein.

Animals↗
PubMed1997

Genetic and antigenic diversity of Ehrlichia chaffeensis: comparative analysis of a novel human strain from Oklahoma and previously isolated strains.

A new Ehrlichia strain, designated as Ehrlichia chaffeensis, Sapulpa strain, was isolated from a patient from Oklahoma with severe ehrlichiosis. Isolation of the ehrlichial pathogen was achieved by inoculating patient blood onto HEL cells and DH82 cells. Antigenic properties of the new isolate were characterized with monoclonal antibodies, homologous patient serum, and polyclonal rabbit serum by Western immunoblotting. The results showed antigenic differences and protein size variation of Sapulpa strain compared with the other 2 strains of E. chaffeensis. Sequencing of the 16S rRNA gene showed 100% identity to that of E. chaffeensis, strain 91HE17. Polymerase chain reaction and sequencing of DNA homologous to the 120-kDa protein gene of E. chaffeensis, Arkansas strain, showed that this gene of Sapulpa strain was smaller than that of Arkansas strain and contained a repeat region with three tandem repeat units.

Animals↗
PubMed1997

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

3T3 Cells↗
PubMed1997

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

Animals↗
PubMed1997

Clonal diversity of primitive human hematopoietic progenitors following retroviral marking and long-term engraftment in immune-deficient mice.

The ultimate goal of human gene therapy in treating hematopoietic disorders is to insert a functional copy of the affected gene into self-renewing stem cells. The engineered pluripotent cells should then provide all lineages of corrected blood cells for the lifetime of the recipient. It is therefore important to develop methods of tracking and studying the progeny of individual human hematopoietic stem cells. Using the technique of single-colony inverse polymerase chain reaction (PCR), we assessed the clonal diversity of marked colony-forming cells that had developed from transduced human hematopoietic progenitors in a long-term xenograft system. The LN retroviral vector, which carries the neo gene, was used to individually mark human CD34+ progenitors. The marked cells were then transplanted into immune-deficient mice for periods of up to 1 year to assess their survival and retention of clonogenic capacity. Following long-term engraftment, bone marrow cells recovered from each mouse were plated in human-specific colony-forming unit (CFU) assay with and without the drug G418, which selects for cells expressing the neo gene. Three weeks later, well-isolated colonies that had grown in G418 were plucked, and PCR for the neo gene was performed to confirm the presence of the vector. Inverse PCR was then performed on neo+ colonies to analyze the integration site of the LN provirus in human DNA. The clonal diversity of G418-resistant (G418R) human CFU recovered from 18 long-term engrafted beige/nude/xid (bnx) mice was assessed. From one to six human hematopoietic precursors had generated all marked colony-forming progenitors (3-39) recovered from the marrow of each animal. To assess the extent of in vitro self-renewal divisions, marrow samples from 22 sets of experiments, with 2-4 mice transplanted in each set, were studied using the single-colony inverse PCR technique. Proviral integrants at identical sites were found in only two mice transplanted with cells transduced in the same flask. The presence of identical integration sites in human progenitors recovered from two mice demonstrated that a long-lived, marked cell had self-renewed in vitro before transplantation and that both daughter cells had retained the capacity to home to the bnx bone marrow and survive for 10 months. Our in vivo xenograft model and the inverse PCR technique have allowed us to identify, trace, and quantitate the clonogenic progeny of primitive human hematopoietic cells for up to 1 year after retroviral-mediated transduction.

Animals↗
PubMed1996

Altered expression of microtubule-associated proteins in cat trochlear motoneurons after peripheral and central lesions of the trochlear nerve.

Neurons lesioned in the peripheral nervous system (PNS) generally regenerate and survive, while neurons lesioned in the central nervous system (CNS) do not regenerate and often die. Investigators have traditionally compared the neuronal responses to PNS and CNS lesions in two separate populations of neurons. In this study, we compared the effects of PNS and CNS lesions on the expression of cytoskeletal proteins in a single neuronal population, the trochlear motoneurons of the cat. The trochlear nerve was lesioned either unilaterally in the PNS or bilaterally in the CNS (within the anterior medullary velum), and animals were allowed to survive 1, 2, or 4 weeks. Brain sections were reacted immunocytochemically using antibodies against microtubule -associated protein-2 (MAP-2) and a phosphorylated isoform of MAP1B, termed MAP1B-P. MAP-2 immunoreactivity (IR) was significantly decreased in the CNS-lesioned trochlear nucleus, compared to the lesioned and the unlesioned trochlear nucleus of PNS-lesioned animals. MAP1B-P IR was significantly increased in PNS- and CNS- lesioned trochlear axons, compared to axons in the unlesioned trochlear nerve of PNS-lesioned animals, and appeared in a small percentage of PNS- and CNS-lesioned cell bodies. These results support the growing body of evidence that MPA-2 can serve as a marker for cells that will eventually die following neuronal insult. The increased immunostaining of MAP1B-P in lesioned axons and its appearance in lesioned cell bodies are characteristic of the immature CNS and may reflect an initial recapitulation of early development, when the levels of this protein are high.

Animals↗
PubMed1996

Neuropeptide Y Y2 receptor-mediated attenuation of neurogenic plasma extravasation acting through pertussis toxin-sensitive mechanisms.

1. The effects of neuropeptide Y (NPY) receptor agonists (administered intravenously) were examined on plasma protein ([125I]-bovine serum albumin) leakage within dura mater evoked by unilateral trigeminal ganglion stimulation (0.6 mA, 5 ms, 5 Hz, 5 min), capsaicin (1 mumol kg-1, i.v.) or substance P (1 nmol kg-1, i.v.) in anaesthetized Sprague-Dawley rats. 2. NPY (EC50: 5.6 nmol kg-1) and NPY fragment 13-36 [NPY (13-36)] (ED50: 4.3 nmol kg-1), an NPY Y2 receptor agonist, dose-dependently attenuated [125I]-bovine serum albumin extravasation from meningeal vessels when administered 10 min prior to electrical stimulation. [Leu31, Pro34]-NPY, an NPY Y1 and Y3 receptor agonist, inhibited the response at a higher dose only (23 nmol kg-1) (P < 0.05). 3. NPY also significantly decreased plasma protein extravasation induced by capsaicin (1 mumol kg-1) but not by substance P (1 nmol kg-1). 4. Pertussis toxin (20 micrograms kg-1, administered intracisternally 48 h prior to stimulation) blocked completely the inhibitory effect of NPY and NPY (13-36) but did not inhibit extravasation alone. 5. We conclude that NPY inhibits neurogenically-mediated plasma protein extravasation acting through presynaptic pertussis toxin-sensitive NPY Y2 receptors, possibly by inhibition of neuropeptide release from perivascular trigeminovascular afferents.

Animals↗
PubMed1996

Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico.

Although Rocky Mountain spotted fever was documented in northern Mexico during the 1940s, spotted fever group (SFG) rickettsioses have subsequently received little attention in Mexico. In this study, sera collected in 1993 from 50 patients from the Mexican states of Yucatan and Jalisco, who were suspected clinically to have dengue fever but had no antibodies to dengue virus, were examined by indirect immunofluorescence for IgM antibodies reactive with Rickettsia rickettsii, R. akari, and R. typhi. Twenty (40%) of the patients' sera contained IgM antibodies to SFG rickettsiae at a titer of 128 or greater. Among five sera reactive only against R. akari, four were from patients in Jalisco where a cluster of cases occurred in June and July. Among five sera reactive only with R. rickettsii, all were from Yucatan patients. Sera of 10 patients contained antibodies reactive with antigens shared by R. rickettsii and R. akari. The clinical signs and symptoms (fever, 100%; myalgia, 95%; headache, 85%; rash, 85%) were similar to those of dengue fever patients identified in this study. However, the incidence of rash was substantially higher than the nondengue, nonrickettsiosis patients. One or more SFG rickettsioses appear to be present in areas of Mexico not previously recognized to harbor these organisms. The etiologic agent or agents are as yet unknown.

Adolescent↗
PubMed1996

5-Carboxamido-tryptamine, CP-122,288 and dihydroergotamine but not sumatriptan, CP-93,129, and serotonin-5-O-carboxymethyl-glycyl -tyrosinamide block dural plasma protein extravasation in knockout mice that lack 5-hydroxytryptamine1B receptors.

We studied the dural plasma protein extravasation response after unilateral electrical stimulation of the trigeminal ganglion in mice lacking serotonin 5-HT1B (5-HT1D beta) receptors by modifying a technique previously described in rats or guinea pigs. We investigated the inhibitory effects of six 5-HT1 receptor agonists in this model: 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), sumatriptan, serotonin-5-O-carboxymethyl-glycyl -tyrosinamide (GTI), 5-methylaminosulfonylmethyl-3-(N-methylpyrrolidin-2R -ylmethyl)-1H-indole (CP-122,288), 5-carboxamido-tryptamine (5-CT), and dihydroergotamine. The plasma extravasation response did not differ between wild-type and mutant after vehicle injection. The potency of sumatriptan, CP-122,288, CP-93,129, and 5-CT in wild-type mice was similar to that previously reported for rats. CP-122,288 (1 nmol kg), 5-CT (1 nmol/kg), and dihydroergotamine (72 nmol/kg) inhibited plasma protein extravasation within dura mater after electrical trigeminal ganglion stimulation in both wild-type and knockout mice, which suggests that these agonists act predominantly via receptors other than 5-HT1B. Unlike the wild-type mice, CP-93,129 (1.4 mumol/kg), a specific 5-HT1B receptor agonist, had no effect in knockout mice. The same held true for sumatriptan (0.7 mumol/kg) and GTI (0.6 mumol/kg). These results suggest that CP-93,129, sumatriptan, and GTI exert their effects via 5-HT1B (5-HT1D beta) receptors in mice.

Animals↗
PubMed1996

Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells.

A persistent obstacle in the use of vector systems for gene therapy has been the inability to attain high-level expression of the target gene in primary cells in vivo. The MFG retroviral vector was designed to yield improved expression over the widely used N2 or LN vectors; however, the molecular basis for this effect has not been examined. Using the human glucocerebrosidase (GC) enzyme as a reporter, we compared expression from the MFG and N2 vector backbones in transduced murine hematopoietic cells after syngeneic bone marrow transplantation. Reporter enzyme activities in primary spleen colonies of transplanted mice were seven-fold higher per vector copy in cells transduced with the (MFG-based) MGC vector than in cells bearing the (N2-based) G2 vector. In spleen colonies harboring the MGC vector, the ratio of spliced to unspliced vector RNA was increased four-fold relative to the G2 vector transcripts in Northern blot analyses. Further analyses indicated that MGC-transduced cells contained five-fold higher levels of spliced RNA per vector copy. Since translation of spliced RNA species (in which the complex secondary structure of the packaging signal has been excised) is likely to proceed with enhanced efficiency, the augmented levels of spliced RNA produced by MFG may represent the key element of increased protein expression from this vector. These findings suggest that the MFG retroviral vector may provide higher level expression of target genes used in human gene therapy.

Animals↗
PubMed1996

Endogenous inhibitors of nitric oxide synthesis and lipid peroxidation in hyperlipidemic rabbits.

AIM: To examine whether the elevation of endogenous NG, NG-dimethylarginine (DMA) content is related to lipid peroxidation in the high lipid-fed rabbit. METHODS: In high lipid diet-fed rabbits, concentrations of serum cholesterol, triglyceride, malondialdehyde (MDA), and DMA were measured, and endothelium-dependent relaxation to acetylcholine (ACh) was tested. RESULTS: After 6-wk on a high lipid-diet, the levels of serum total cholesterol, triglyceride, MDA, and DMA were increased vs those in control group (MDA was 2.88 +/- s 0.20 vs 1.54 +/- 0.13 nmol.L-1, P < 0.01 and DMA was 1.51 +/- 0.07 vs 0.75 +/- 0.13 mumol.L-1, P < 0.01), while the endothelium-dependent vasodilation in the isolated thoracic aorta was impaired (the maximal response to ACh was 45.59 +/- 3.10 vs 76.93 +/- 5.68%). Supplementation with vitamin E decreased MDA and DMA content and improved the endothelium-dependent vasodilation. CONCLUSIONS: An increase in serum concentration of DMA may be secondary to the elevation of lipid peroxides in the hyperlipidemic rabbit.

Animals↗
PubMed1995

Retroviral-mediated gene expression in human myelomonocytic cells: a comparison of hematopoietic cell promoters to viral promoters.

Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.

Antigens, CD34↗