Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34+ cells emphasize limited transduction at low receptor levels

Abstract

Successful gene therapy for the treatment of heritable or acquired diseases typically requires high efficiency gene transfer and sustained transgene expression. Indirect evidence on the basis of RNA analysis and in vivo competitive repopulation experiments in animal models suggests a correlation between transduction efficiency and the abundance of retrovirus receptors on the hematopoietic target cell. However, transduction by oncoretroviral vectors is also subject to other factors such as target cell cycle status and the composition of the virus-containing medium, making it difficult to determine the level of receptor expression required for efficient transduction. In the present study we investigated the impact of receptor expression level on transduction by a vector with a gibbon ape leukemia virus (GALV) envelope protein in a tetracycline-inducible tissue culture model that allowed for the cell cycle-independent, regulated expression of the GALV receptor (Pit1) in otherwise non-susceptible NIH 3T3 cells. Up-regulation of receptor RNA expression by 4.5-fold resulted in a mean 150-fold increase in transduction efficiency. We then analyzed cell surface expression of the Pit1 receptor using a fusion protein consisting of GALV SU portion of the viral envelope protein linked to the human IgG Fc. These experiments showed that tetracycline-regulated receptor induction resulted in a dose-dependent increase in binding of fusion protein. At maximum induction fusion protein binding increased up to five-fold which paralleled the increase in RNA expression, and correlated with the improved transduction efficiency. Finally, studies of pseudotype-specific fusion protein binding to human CD34-enriched cells revealed increased expression of retrovirus receptors after cytokine stimulation, although overall receptor expression in CD34+cells remained lower than in fibroblast cell lines efficiently transduced by amphotropic and GALV vectors.

Introduction

Murine leukemia virus (MLV)-based retroviral vectors have historically been the preferred method for gene transfer into hematopoietic cells. Major disadvantages of current oncoretroviral vectors derived from MLV include the inability to infect nondividing cells and the need for a specific surface receptor on the target cell.123 Binding and internalization of a packaged vector is dependent on receptor availability in target tissues.4 Retrovirus packaging cell lines can be made using different env genes, most commonly targeting amphotropic (Pit2, also called Ram1 or G1vr2), or gibbon ape leukemia virus (Pit1, also called G1vr1) receptors.56

A number of studies have noted that the abundance of a specific receptor correlates with the efficiency of retroviral transduction.78910 These studies analyzed receptor expression by quantification of mRNA, not allowing for post-transcriptional regulation of receptor expression, or secondary effects of surface receptor density. Others have used an indirect assay with antibody against cell-bound envelope protein.1112 To evaluate more accurately the cell surface expression of retroviral receptors, we have designed fusion proteins that join the amino-terminal SU portion of the envelope protein to the Fc region of a human IgG 1 molecule. In a previous study we performed analysis of receptor expression by flow cytometric study of fusion protein cell surface binding, and enumerated receptors by Scatchard analysis.13 We demonstrated that efficient transduction of 208F rat fibroblasts by an amphotropic pseudotype retrovirus vector was dependent on high-level expression of the receptor Pit2, and correlated with receptor number and density. Stable overexpression of the receptor in that study precluded analysis of how low receptor levels impact transduction efficiency, a question directly relevant to the transduction of hematopoietic cells.

The current study examined the correlation of low level Pit1 expression and target cell transduction efficiency. The tetracycline-controlled gene expression system is based on tetracycline-resistance operon tet from E. coli transposon 10. In this system, the tetracycline-controlled transactivator protein (tTA) is composed of the tet repressor (tetR) and the activating domain of the viral protein VP16 of herpes simplex virus which strongly activates transcription.1415 The transcription of the gene of interest, ie Pit1, is controlled by a minimal promoter from human cytomegalovirus (hCMV) fused to the tet operator sequence. The tTA binds to the tet operator sequences in the absence of tetracycline resulting in repression of transcription in the presence of tetracycline.

Using a fusion protein with Pit1 receptor-binding SU domain (GSU-hFc), we demonstrate that induction of Pit1 receptor expression is directly correlated with increased transduction efficiency in otherwise non-susceptible NIH 3T3 cells. In addition, we confirm that CD34+ cells express comparatively low levels of the retrovirus receptors Pit1 and Pit2 compared with the fibroblast cell lines analyzed, in part explaining their resistance to efficient retroviral transduction.

Results

Induction of Pit1 receptor expression and transduction efficiency

We have studied the influence of retrovirus receptor expression on gene transfer efficiency by expressing Pit1 in NIH 3T3 TK cells using a tetracycline-controlled gene expression system. AA4T/Pit1 cells, containing the tetracycline-control plasmid pUHD15-1 and the response plasmid pUHD10-3 expressing Pit1 under the control of a tetracyline-responsive promoter, were cultured in the presence of different concentrations of tetracycline. Northern blot analysis of RNA from AA4T/Pit1 cells cultured in the presence of tetracycline concentrations ranging from 102 to 10−4 μg/ml revealed that receptor RNA expression was very sensitive to the addition of tetracycline and was down-regulated up to 4.5-fold in a dose-dependent manner (Figure 1a). The figure shows the expected larger murine Pit1 RNA, the smaller tetracycline-repressible human Pit1 RNA below, and the human Pit1 used in stably overexpressing NIH 3T3 cells (top right hand).

Figure 1
figure1

Pit1 RNA induction and transduction in tetracycline-regulated NIH 3T3 cells. (a) Otherwise non-susceptible NIH 3T3 TK cells were stably transfected with an inducible Pit1 expression system. RNA was harvested from cells grown in the presence of tetracycline at concentrations indicated, or in its absence. The figure shows hybridization of a human Pit1 probe to the tetracycline-inducible human Pit1 (hPit1, small fragment), the retrovirally expressed human Pit1 (hPit1, large fragment) in the retrovirus vector LPit1SHD, and cross-hybridization of the probe to the endogenous murine Pit1 analogue (mPit1). Human control cell lines K562, KG1 and IB3 show Pit1 expression in greater abundance than inducible AA4T/Pit1 cells. A faint hybridization signal that runs above the murine Pit1 RNA species probably represents as alternatively spliced message with a different polyadenylation tail transcribed from the inducible hPit1 expression plasmid. The figure shows a representative experiment. (b) Transduction efficiency of NIH 3T3 mouse fibroblasts (AA4T/Pit1) with vector LAPSN(PG13). Cells were plated at 105 cells per 6-cm plate and tetracycline was added to medium at the concentrations indicated. Cells were transduced 12 h later with vector LAPSN(PG13) and transduction efficiency was assessed in focus-forming units (FFU) per ml vector by counting AP+FFU. The figure shows means from three independent experiments with error bars depicting standard deviations. (c) Correlation of induced human Pit1 mRNA and transduction efficiency with an alkaline phosphatase encoding retrovirus vector. RNA was quantified by phosphoimager in volume units, while AP+FFU were enumerated as outlined above. The figure depicts RNA induction data from a representative experiment, which was repeated a second time with similar results.

To study the impact of receptor RNA expression level on transduction efficiency, we transduced NIH 3T3 cells with GALV pseudotype retroviral vector LAPSN(PG13) and scored successful infection by enumeration of AP-positive focus-forming units (AP+FFU). Figure 1b summarizes these experiments and demonstrates the predicted inverse correlation between tetracycline concentration in the culture medium and the number of AP+FFU. This confirms a 150-fold mean increase in transduction efficiency in this model mediated by tetracycline withdrawal, and the resultant induction of Pit1 RNA expression. Maximum inhibition of receptor expression was achieved at concentrations >1 μg/ml in the medium (Figure 1b). Expression of Pit1 RNA extracted from cells grown at the different tetracycline concentrations correlated well with the lower transduction rates observed with the addition of tetracycline (Figure 1c). Interestingly, changes in transduction efficiency were more pronounced at overall low levels of receptor in the left hand part of the curve (Figure 1c).

The sensitivity to tetracycline induced Pit1 down-regulation and thereby stability of expression was investigated in two independent experiments measuring the transduction efficiency of AA4T/Pit cells at serial time-points after addition of tetracycline (10 μg/ml). The results demonstrate that receptor expression is almost entirely down-regulated between 6 and 12 h after addition of drug (Figure 2).

Figure 2
figure2

Time-course of Pit1 down-regulation measured by transduction efficiency of NIH 3T3 mouse fibroblasts (AA4T/Pit1) with vector LAPSN(PG13). Cells were plated at 105 cells per 6-cm plate, and transduced with vector LAPSN(PG13) at time-points indicated after removal of tetracycline from media. Transduction efficiency was assessed in focus-forming units (FFU) per ml vector by counting AP+FFU. The figure shows means and error bars based on two independent experiments.

To determine whether modulation of Pit1 by tetracycline would alter the growth characteristics of AA4T/Pit1 cells, we performed cell cycle analysis following exposure of these cells to a five-log range of tetracycline concentration. MULTICYCLE analysis of cell cycle phases revealed no differences (data not shown).

Binding of GSU-hFc to Pit1

We have previously documented the utility of pseudotype-specific fusion proteins to quantify the cell surface expression of retroviral receptors.13 We demonstrated that cell surface binding of amphotropic fusion protein to retroviral receptors correlated with transduction efficiency of rat fibroblasts by an amphotropic pseudotype vector.

To demonstrate that the increase in gene transfer efficiency in the tetracycline-inducible system is correlated not only with increased Pit1 RNA expression, but indeed with increased cell surface receptor expression, we performed flow cytometric binding studies using the GALV pseudotype fusion protein (GSU-hFc). Figure 3 confirms that GSU-hFc does not bind to parental NIH 3T3 cells but does bind to NIH 3T3 cells expressing Pit1. Expression under the control of the tetracycline-inducible system was lower than expression from a retroviral LTR such as in pLPit1SN. This finding correlated with the lower Pit1 RNA expression in inducible AA4T/Pit1 cells compared with NIH 3T3/LPit1SHD cells (Figure 1). GSU-hFc binding increased predictably over a range of tetracycline concentrations up to five-fold and in parallel with the induction of mRNA (Figure 4). Finally, using AA4T/Pit1 cells at selected tetracycline concentrations, or stable receptor overexpression on NIH 3T3/LPit1SHD cells, the binding of fusion protein and vector transduction efficiency, as functions of receptor expression, correlated. While low levels of receptor prevented better resolution of fusion protein binding using a FACS assay, Figure 5 suggests a linear relationship at higher levels of receptor expression.

Figure 3
figure3

Histogram analysis depicting the impact of Pit1 receptor expression on GALV pseudotype fusion protein binding to cells. Binding was studied in unmodified NIH 3T3 cells, NIH 3T3 cells stably transfected with an inducible expression cassette (AA4T/Pit1) at different tetracycline concentrations, and NIH 3T3 cells overexpressing the receptor Pit1 in a retroviral construct (NIH 3T3/LPit1SHD). Flow-cytometric analysis was carried out following incubation with fusion protein GSU-hFc or IgG1 isotype followed by incubation with PE-fluorescent secondary anti-mouse IgG1 antibody. The y-axis depicts median fluorescence units as calculated by Cellquest (Becton Dickinson) software. Isotype staining was similar to fusion protein staining of NIH 3T3 cells and is not depicted.

Figure 4
figure4

RNA induction and GSU-hFc binding using inducible AA4T/Pit1 cells over a range of tetracycline concentrations. Cells were plated at 105 cells per 6-cm plate and tetracycline was added to medium at the concentration indicated. Cells were transduced with vector LAPSN(PG13). Aliqouts of cells were analyzed by flow cytometry following incubation with fusion protein GSU-hFc or IgG1 isotype followed by PE-fluorescent secondary anti-mouse IgG1 antibody. The right hand y-axis depicts median fluorescence units as calculated by Cellquest (Becton Dickinson) software. In parallel RNA was extracted from aliquous of cells identically cultured, and quantified by Northern blot with subsequent Phosphoimager analysis as described. Volume units calculated by ImageQuant software are depicted on the left-hand y-axis. Results of fusion protein binding reflect average values and errors based on two independent experiments. Northern analysis was performed twice with similar kinetics The plot shows representative results from experiment 1.

Figure 5
figure5

Correlation of transduction efficiency and fusion protein binding as functions of receptor expression. Binding was studied using AA4T/Pit1 cells in two independent experiments at selected tetracycline concentrations of 0 and 10 μg/ml, and in a single experiment using stable receptor overexpression on NIH3T3/LPit1SHD cells. Transduction efficiency and fusion protein binding are scored as previously described. The figure integrates data points from two independent experiments.

Cytokine-induced expression of Pit1 and Pit2 on human CD34+ cells

We next wished to analyze the expression of Pit1 and Pit2 retrovirus receptors on human hematopoietic stem/progenitor cells. In initial studies, buffy coat cells from volunteer bone marrow donors were isolated and enriched for CD34-expressing cells by immunomagnetic selection. These cells were then analyzed for expression of Pit1 and Pit2 receptor by incubation with fusion protein GSU-hFc, or ASU-hFc and secondary PE-labeling monoclonal antibody. Results after 24 h of incubation in the presence of IL-3, IL-6, and SCF showed only a small increase in fusion protein binding over that of isotype or secondary antibody indicating low-level expression of receptor. Incubation of cells in the presence of cytokines for 72 h, analogous to our primate transduction protocols, demonstrated a substantial increase in fusion protein binding (Figure 6a). This suggests up-regulation of expression of Pit1 and Pit2 retrovirus receptors on the surface of CD34+ cells during continued stimulation of cells. Culture of cells beyond 3, and up to 6 days did not result in further up-regulation of receptor expression (data not shown). However, compared with cell lines overexpressing the corresponding receptor (NIH 3T3/LPit1SHD, 208F/LPit2SN), these levels were relatively low (Figure 6a). This may in part explain limited gene transfer efficiencies in hematopoietic progenitor/ stem cells.

Figure 6
figure6

Fusion protein binding to human CD34-enriched cells. (a) Binding to CD34+ cells and fibroblast cell lines stably overexpressing the receptors Pit1 and Pit2 was studied using the fusion proteins GSU-hFc and ASU-hFc, respectively. Harvested CD34+ cells were enriched as described in Materials and methods and cultured in the presence of IL-3, IL-6 and SCF for 24 or 72 h. Fibroblasts were detached from dishes by trypsinization and washed in PBS/2% FBS. Flow cytometric analysis was carried out following incubation with fusion protein GSU-hFc, ASU-hFc, or IgG1 isotype followed by PE-fluorescent secondary anti-mouse IgG1 antibody. The x-axis depicts the fold increase in median fluorescence units as calculated by Cellquest (Becton Dickinson) software. The figure summarizes results from three independent experiments. Background binding of isotype control was similar in fibroblast and CD34+ cells and is not depicted. (b) Comparative fusion protein binding to human CD34+ cells using amphotropic (ASU- hFc) and GALV (GSU-hFc) envelope fusion proteins. Cells were cultured in the presence of IL-3, IL-6 and SCF for 72 h. Flow cytometric analysis was carried out following incubation with fusion proteins ASU-hFc, GSU-hFc, or IgG1 isotype followed by PE-labeled secondary anti-mouse IgG1 antibody. Results depicted are representative of four independent experiments.

Finally, we investigated the relative binding of amphotropic and GALV pseudotype fusion proteins on CD34-enriched cells after 72 h of incubation in cytokine-supplemented medium (IL-3, IL-6 and SCF) (Figure 6b). In four separate experiments (three using bone marrow, one using PBSC) there was a trend toward increased GSU-hFc over ASU-hFc binding, indicating increased Pit1 expression in comparison to Pit2 expression on these cells.

Discussion

Investigations into limitations to retrovirus transduction due to low receptor levels are directly relevant to clinical gene therapy efforts. In gene transfer studies using GALV and amphotropic pseudotype vector, increased transduction rates correlated with the higher expression of Pit1 compared with Pit2 receptor RNA in human and non-human primate hematopoietic target cells.91617

To study directly the impact of changes in cell surface receptor abundance at low overall receptor levels, we developed a tissue culture model allowing the tetracycline-inducible down-regulation of a heterologous retrovirus receptor in NIH 3T3 cells. This cell line is normally not susceptible to transduction by GALV vectors, and therefore no interference from endogenous receptor had to be adjusted for in our experiments.

Using a fusion protein containing the GALV SU domain, we observed decreased surface receptor binding that was mediated by down-regulated Pit1 expression on NIH 3T3 TK cells within 12 h of tetracyline induction. Down-regulation functioned in a tetracyline concentration-dependent manner and correlated with transduction efficiency and mRNA induction of our target cell line. This indicates that transduction efficiency in target cells can be optimized by increasing receptor expression or, conversely, by transducing cells with a vector pseudotyped to target the receptor more abundantly expressed in the tissues of interest. Optimum Pit1 receptor expression, and thus transduction efficiency, is probably higher than achieved in our tetracycline-inducible system. Indeed, in this model, fusion protein binding as a measure of Pit1 expression was limited to a 1.5-log fluorescence increase over isotype, as compared with 2.5-log fluorescence separation using stably overexpressed Pit1 transcribed from a retroviral LTR. As demonstrated in Figures 3 and 5, at low overall levels of receptor, small differences in expression translate into major increases in transduction efficiency, an effect that becomes less pronounced at higher overall receptor levels. This may indicate that receptor surface density reflected by fusion protein binding may increasingly impede a further linear increase in target cell transduction at high receptor levels.

Others have previously shown, and here we have confirmed that inducing hematopoietic cells into cell cycle with cytokine support increases transduction efficiency, probably in part by up-regulating retrovirus receptor expression of CD34+ target cells.8111218192021 When we used the fusion proteins described here to study retrovirus receptor expression of human marrow and PBSC CD34+ cells, we found more binding by GALV than amphotropic pseudotype fusion protein, confirming our previous mRNA-based findings.7 If our observations in the tetracycline-inducible system can be extrapolated to the transduction of CD34+ hematopoietic cells, the relatively small increase in GALV over amphotropic fusion protein binding at low receptor levels may imply significant differences in target cell transduction. This is consistent with findings in our baboon repopulating studies demonstrating increased marking from GALV versus amphotropic pseudotype vectors.7

In conclusion, our results indicate that the level of expression of the appropriate retroviral receptor is an important factor in predicting oncoretroviral transduction efficiency of target cells, and may be preferentially limiting at low overall levels of receptor expression. As a means to optimize gene transfer into hematopoietic tissues with oncoretroviral vectors for clinical trials, targeting the virus receptor that is expressed most abundantly will be crucial.

Materials and methods

Construction of Pit1-expression plasmids

The regulator plasmid encoding tTA, pUHD15–1, and the response plasmid containing a tTA-dependent promoter in front of the reporter gene of interest, pUHD10–3, have been described1415 and were provided by H Bujard (Zentrum für Molekulare Biologie, Universitaet Heidelberg, Germany). The cDNA for Pit1, the gibbon ape leukemia virus receptor, was obtained from B O'Hara (Department of Molecular and Structural Biology, University of Aarhus, Denmark) and was cloned as a 2.2 kb EcoRI–BamHI fragment into the multiple cloning site of pUHD10–3 to make pHUD10–3hPit1. The retroviral vector pLXSHD22 was used to make pLPit1SHD by insertion of the human Pit1 sequence.

Construction of stably transfected cell lines

NIH 3T3 thymidine kinase-negative (TK) cells were first contransfected with the pUHD15–1 and a neo-containing plasmid (Rosa βgeo provided by P Soriano, Fred Hutchinson, Cancer Research Center, Seattle, WA, USA). G418-resistant clones were isolated and screened for their ability to induce luciferase expression in plasmid pUHC13–3 (provided by H Bujard). The clone with the highest induction of luciferase expression was named AA4T and was used for introduction of the response plasmid containing Pit1. AA4T cells were cotransfected with a plasmid containing the HSVtk gene, cloned as a BamHI fragment into the BamHI site of pBR322, and pUHD10–3hPit1. Stably transfected clones were isolated in HAT medium (100 μm hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, and 3 mM glycine). Clones were screened for their susceptibility to retrovirus transduction with a GALV-pseudotype vector LAPSN(PG13). The best clone, c19, was used for the receptor induction experiments and named AA4T/Pit1.

Cell lines and transductions

NIH 3T3 TK cells and packaging cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/l of glucose, 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco BRL, Grand Island, NY, USA). Retrovirus-containing medium was harvested from PG13 cells transduced with the LAPSN vector6 (PG13/LAPSN), filtered and stored at −80°C. Transduction of AA4T cells was performed with 1 ml of virus containing medium in the presence of 4 μg/ml Polybrene. 208F rat fibroblasts were transduced with LPit2SN vector to generate the Pit2-overexpressing cell line 208F/LPit2SN.

Expression analysis – histochemical staining of alkaline phosphatase (AP)

This has been described.23 Briefly, cells were fixed in phosphate-buffered saline (PBS) containing 0.5% glutaraldehyde for 15 min, washed twice in PBS and incubated at 65°C for 30 min to eliminate endogenous AP. For AP staining, cells were incubated overnight at room temperature in 100 mM Tris (pH 8.5), 100 mM NaCl, 50 mM MgCl2, 1 mg of nitro blue tetrazolium per milliliter, 0.1 mg of 5-bromo-4-chloro-3-indolyl phosphate per milliliter.

Expression analysis – flow cytometric analysis

Fusion protein binding was assessed by flow cytometric analysis. Cells were detached by trypsinization, or with 0.5 mM EDTA, and were incubated with either the amphotropic SU fusion protein, GALV SU fusion protein, or an irrelevant polyclonal human IgG Ab (DAKO, Glostrup, Denmark) for 60 min at 4°C. Cells were then washed twice, incubated with a PE-conjugated F(ab)2 fragment from a rabbit antibody directed against human Fc (DAKO) for 30 min at 4°C, washed twice, and suspended in PBS containing 2 μg propidium iodide per milliliter. FACS analysis was carried out on a Calibur (Becton Dickinson, San Jose, CA, USA). Viability was determined by standard propidium iodide staining.

Construction and purification of GALV- and 4070A SU-immunoadhesin (GSU-hFc, ASU-hFc)

GSU-hFc was constructed by amplifying the first 437 amino acids of the SU region of the SEATO strain of GALV and introducing a SalI site at the 3′ end. The PCR product was cloned into the pCR3.1-Uni vector (Invitrogen, Carlsbad, CA, USA). pCR3.1-Uni was cut with SalI and NotI to introduce the human Fc portion of IgG1 (kindly provided by D Cosman, Immunex, Seattle, WA, USA). For initial experiments, the pCR3.1-Uni vector was used for expression of the GSU-hFc fusion protein. More recently, we have used the pCMVβ construct from Clontech, which contains a CMV promoter upstream of the GSU-hFc gene, an SV40 intron, and a SV40 polyadenylation signal, for expression of our fusion protein (Clontech Laboratories, Palo Alto, CA, USA). For this purpose, a HindIII–XhoI fragment from pCR3.1-Uni was subcloned into Bluescript (Clontech) and a NotI–NotI fragment containing the GSU-hFc fragment was cloned into the pCMVβ vector. Amphotropic fusion protein, ASU-hFc, was constructed in analogous fashion with a DNA fragment that encoded the SU portion of the amphotropic 4070A Env protein, minus the carboxy-terminal nine amino acids, linked to the human IgG–Fc protein fragment lacking the amino-terminal three amino acids after the Fc cleavage site. This DNA was cloned in place of β-gal cDNA in pCMVβ.

Transfection and purification of ASU-hFc and GSU-hFc

For large-scale production of fusion proteins, the expression plasmids were transfected by CaPO4 coprecipitation into 293 cells plated at 70% confluence in 15-cm dishes 1 day earlier. The fusion protein was harvested in culture medium containing low IgG serum (Gemini Bioproducts, Calabasas, CA, USA) at 24 and 48 h after transfection, and filtered (0.45 μm pore size) to remove particles and debris. The harvested protein was passed over a protein A column (Pharmacia, Peapack, NJ, USA). The column was washed with 20 mM NaPO4 (pH 7) for 15 min and the fusion proteins were eluated in 0.1 M citric acid (pH 3) and immediately neutralized in 10× PBS (pH 8.5). The purified protein was concentrated with a centricon membrane (50 000 Kd cut-off). The concentration of the fusion proteins was determined by spectophotometry at 260/280 nM, and confirmed by Coomassie blue staining in comparison with bovine serum albumin.

Northern analysis

RNA from cell lines was isolated using RNAzol (MRC, Cincinnati, OH, USA) according to the manufacturer's instructions. Northern blots were hybridized with a 488 bp DraI–XhoI fragment from the Pit1 sequence.

Enrichment and culture of CD34 human progenitor cells

Bone marrow from normal donors was obtained after informed consent according to IRB approved guidelines. Cells were harvested in preservative-free heparin and resuspended in phosphate-buffered saline. Buffy coat cells were labelled with IgM monoclonal antibody 12–8 (CD34) at 4°C for 30 min, washed, incubated with rat monoclonal anti-mouse IgM microbeads (Miltenyi Biotec, Auburn, CA, USA) for 30 min at 4°C, washed, and then separated using an immunomagnetic column technique (Miltenyi Biotec) according to the manufacturer's instructions. The purity of CD34-enriched cells was between 65 and 85%. Peripheral blood stem cells from a donor were processed identically. Cells were cultured for 72 h in Iscove's modified Dulbecco's medium supplemented with 12.5% horse serum (Gibco BRL), 12.5% FBS (Gibco BRL) 10−6 M hydrocortisone (Sigma Chemical Co, St Louis, MO, USA), 0.1 mM 2-mercaptoethanol (Sigma), penicillin, streptomycin, and 2 mM glutamine. In addition, IL-3, IL-6 and stem cell factor were added at 50 ng/ml each.

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Acknowledgements

This work was supported by grants DK47754 and HL36444 from the National Institutes of Health. HPK is a Markey Molecular Medicine Investigator.

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Correspondence to H-P Kiem.

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Kurre, P., Morris, J., Miller, A. et al. Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34+ cells emphasize limited transduction at low receptor levels. Gene Ther 8, 593–599 (2001). https://doi.org/10.1038/sj.gt.3301438

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Keywords

  • transduction
  • fusion protein
  • retrovirus receptor
  • Pit1
  • Pit2

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