The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3′ enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin− bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19+ cells, but not by CD3+, CD11b+, CD11c+ or Gr-1+ cells. CD19+ cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19+ cells, while eGFP was expressed by both spleen focus-forming virus and cytomegalovirus constitutive promoters in CD19+ and CD3+ lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.
Introduction of genes into haematopoietic cell lineages potentially offers a well-controlled means to study the function of specific genes and their products in experimental immunology. Additionally, in clinical medicine, gene therapy approaches with haematopoietic cells or their stem cell precursors as the target, can be used for treating a number of conditions including immunodeficiencies,1, 2 malignancies and lymphoproliferative disorders.3 It is also anticipated that in vivo gene delivery will be useful for manipulating specific immune responses such as the induction of tolerance in autoimmunity and transplantation,4, 5 and immunizations using DNA vaccines.6
Lentivirus-derived vectors constitute an efficient and potentially very safe delivery vehicle for therapeutic genes and are preferable to γ-retroviral vectors as they can target and integrate into non-dividing cells, such as haematopoietic stem cells, with high efficiency.7 As a consequence, clinical trials for treatment of human immunodeficiency virus (HIV)8 and β-thalassaemia9 have been performed using lentiviral vector delivery systems with general viral promoters driving ubiquitous expression in all cell types.
In experimental investigations cell-specific promoters are powerful tools for detailed studies of gene functions in specific cells, for example in the immune system. In many intended clinical applications it is necessary to use cell-specific promoters, for several reasons. First, the function of a certain gene product often needs to be restricted to a particular cell lineage to achieve the desired effect; second, expression of the gene in other cell types may have detrimental effects and third, integration of tissue- or cell-specific promoters into patient cells may be safer and less likely to result in endogenous oncogene transcriptional activation. Previous cell-specific lentiviral promoters have been shown to result in erythroid-,10 T lymphoid-,11, 12 and general antigen-presenting cell-specific expression.13 For B cells, previously used retro/lentiviral promoters have included an immunoglobulin (Ig) heavy-chain enhancer in combination with a phosphoglycerate kinase or cytomegalovirus (CMV) promoter, to increase expression,14 a CD19 gene promoter to drive expression of a marker gene in mice using a retroviral vector15 and in human B cells using a lentiviral vector.16 An Ig kappa (Igk) light chain promoter and enhancer has previously been described as a useful B cell-specific promoter in transgenic mice for expression of the haemagglutinin gene of influenza virus.17 Furthermore, an Ig heavy-chain promoter/enhancer has been previously evaluated in a myeloma cell line.18 Igk light chain promoters in a retro/lentiviral context have not been evaluated as far as we are aware.
In this study we investigated the ability of a mouse Igk light chain gene promoter in association with two mouse Ig heavy-chain gene enhancers to provide B cell-specific expression.
An Igk light chain gene promoter19 was cloned together with the 3′ enhancer20 and the intron enhancer21 from a mouse Ig heavy-chain gene (Igk-E). The Igk-E promoter was cloned into a second-generation, self-inactivating (SIN) HIV-1-based lentiviral vector containing both a central polypurine tract (cPPT) and the woodchuck posttranscriptional regulatory element (WPRE), and used to drive expression of the enhanced green fluorescent protein (eGFP) reporter gene (LNT-Igk-E-eGFP). A lentiviral construct with eGFP expression driven by the spleen focus-forming virus (SFFV) promoter was used as a control (LNT-SFFV-eGFP) (Figure 1a).22
To test the specificity of the Igk-E promoter in murine cell lines, A20 B-cell lymphoma, X16C8.5 B-cell lymphoma, 3T3 fibroblast, HCQ4 T-cell hybridoma and bEnd3 endothelial cells were transduced with either the Igk-E-eGFP or SFFV-eGFP lentivirus. eGFP expression was analysed by flow cytometry (fluorescence-activated cell sorting, FACS) 4 days after transduction at identical multiplicity of infection (MOI), as assessed on A20 cells. The Igk-E-eGFP and SFFV-eGFP constructs were also assessed by quantitative PCR, at several identical MOIs, after transduction of A20 cells resulting in an average of 1.35 (range: 1.27–1.42) times the number of Igk-E-eGFP copies per cell as compared to SFFV-eGFP copy numbers (averages of two PCR in triplicate). This therefore showed that it is very unlikely that a bias in copy numbers between the constructs could explain the different patterns of expression in the cell lines. All cell lines expressed eGFP when transduced with SFFV-eGFP lentivirus, although with different transduction efficiencies (47–99% eGFP positivity). However, only the B-cell lymphoma lines, A20 and X16C8.5, expressed eGFP when transduced with Igk-E-eGFP lentivirus. There was no leakage of eGFP expression in murine fibroblasts, T-cell hybridomas or endothelial cells, when transduced with Igk-E-eGFP lentivirus. This indicates that efficient expression from the Igk-E promoter is exclusive to murine B cells (Figure 1b).
Lentiviral transduction of primary murine splenocytes has been reported in both activated (with lipopolysaccharide (LPS)) and non-activated cells.23, 24, 25 To ensure the Igk-E-eGFP lentivirus could transduce murine splenocytes, CD19+ splenocytes from naive mice were either activated (pre-activated or co-activated) and cultured with Igk-E-eGFP lentivirus, or not-activated and cultured with Igk-E-eGFP lentivirus alone. CD19+ B cells from co-activated and pre-activated cells cultures contained 20 and 44% eGFP+ cells, respectively (Figures 2e, f, i and j). All GFP+ cells expressed the co-stimulatory molecule CD86. In the cultures that were not activated with LPS, there were no eGFP+ cells (Figure 2b), perhaps partly due to the poor ability of CD19+ cells to survive in culture for the required time period (compare Figure 2b; 44% CD19+ with Figures 2e and i; 92 and 93% CD19+, respectively). These data indicate that Igk-E-eGFP lentivirus can transduce primary activated murine B cells with considerable efficiency.
Igk light chains, complexed with Ig heavy chains, are first expressed on the cell surface of pre-B cells in fetal liver and bone marrow.26 This suggests the Igk promoter acts early on during B-cell development. To examine whether the Igk-E promoter is leaky in other haematopoietic cells lineages in the developing lymphoid and myeloid compartments, mice were reconstituted with Igk-E-eGFP-transduced bone marrow. Haematopoietic stem cells (Lin−) were transduced with Igk-E-eGFP or SFFV-eGFP lentivirus ex vivo in the presence of cytokines and 24 h later, the transduced cells injected into lethally irradiated syngeneic recipients. An aliquot of the cells were analysed for viral integration by quantitative PCR resulting in SFFV-eGFP; 8.4 copies per cell and Igk-E-eGFP; 2.6 copies per cell, showing that similar copy numbers of the constructs were engrafted in the two experimental groups. Tissues were harvested 16 weeks after reconstitution and analysed by flow cytometry. The reconstitution experiment was performed twice with similar results and data from experiment 2 are presented here (experiment 1: Igk-E-eGFP n=3, SFFV-eGFP n=4, experiment 2: Igk-E-eGFP n=5, SFFV-eGFP n=4).
There was no difference in cell number between the two groups in all lymphoid tissues analysed. Lineage analysis of the total engraftment showed no difference between proportions of individual lymphoid and myeloid cell types indicating no promoter-specific reconstitution bias (data not shown).
eGFP+ cells were found in lymphoid tissues of all reconstituted mice, except in one mouse from the SFFV group and in one mouse from the IgK group that did not engraft. SFFV-eGFP Lin−-cell recipients had eGFP+ cells in primary and secondary lymphoid organs, while Igk-E-eGFP Lin− cell-reconstituted mice had eGFP+ cells in all lymphoid tissues which contain circulating B cells (Figure 3a).
The specificity of the Igk-E promoter was determined by lineage analysis of the eGFP+ cells using three-colour flow cytometry in bone marrow and spleen. Representative plots of splenocytes from each test group of animals are shown in Figure 3b and a compilation of data sets from all animals summarized in Figure 3c. In the bone marrow of SFFV-eGFP Lin− cell-reconstituted mice, the majority of eGFP+ cells were CD11b+ (60%±4.5) indicative of macrophages and granulocytes. The remaining eGFP+ cells were comprised of CD19+ B-1 cells (22%±4.2) and CD3+ T cells (11.2%±1.8). In mice reconstituted with Igk-E-eGFP Lin− cells, the majority of eGFP+ bone marrow cells were CD19+ (72.1%±2.5). The remaining eGFP+ cells comprised small populations of CD3+ (5.1%±1.2) and CD11b+ cells (8.4%±2.4), which may be attributed to non-specific binding by FACS analysis Ab. In the spleen, the eGFP+ cells were predominantly CD19+ in both groups of animals (SFFV-eGFP—70.5%±5.4: Igk-E-eGFP—89.4%±2.3). A substantial proportion of eGFP+ cells in SFFV-eGFP Lin− cell-reconstituted mice were CD3+ or CD11b+ cells (24.5%±2.7 and 11.3%±5.5, respectively), while eGFP+ splenocytes from Igk-E-eGFP Lin− cell-reconstituted mice showed minimal staining in other lineages (less than 5% CD3+, CD11b+, Gr-1+ or CD11c+). As expected since the thymus contains very few B cells, no eGFP+ cells were detected in the thymus of Igk-E-eGFP Lin− cell-reconstituted mice, while eGFP was detected in all stages of thymocyte maturation in thymi from SFFV-eGFP Lin− cell-reconstituted animals and in thymic epithelium (data not shown).
Further analysis of splenic B-cell populations showed eGFP+ cells were both naive marginal zone B cells and mature follicular B cells (Figures 3b and d). Marginal zone B cells (CD21hiCD23int) are recognized to be non-recirculating and thus permanent residents of the spleen, while follicular B cells (CD21intCD23hi) freely move from the spleen to other secondary lymphoid tissues. CD21 and CD23 can also be expressed on follicular dendritic cells, however the eGFP+ splenic population from Igk-E-eGFP Lin- cell-reconstituted mice contained minimal CD11c+ cells. Of the follicular B-cell population, both IgDhiIgMlo and IgDhiIgMhi follicular B cells were detected in the eGFP+ population, thus further demonstrating the specificity of the Igk-E promoter (data not shown). We next analysed the numbers of eGFP+ cells within the Igk+ and Ig λ+ B-cell compartments in the spleen. The ratios of eGFP+ Igk cells versus eGFP+ Igλ cells in the Igk-E-eGFP as compared to the SFFV-eGFP mice were not significantly different (Figure 3e). However, Igλ cells from both experimental groups were consistently expressing less eGFP then the corresponding Igk cells from respective groups.
The lower eGFP expression in Igk-E-eGFP Lin− reconstituted-mice as compared to that of SFFV-eGFP Lin−-reconstituted mice was addressed by determining vector copy number in bone marrow and thymus. The average vector copy number for Igk-eGFP Lin−-reconstituted mice was significantly lower than that of SFFV-eGFP Lin−-reconstituted mice in both tissues (SFFV-eGFP bone marrow; 4, range: 3–8 copies per cell; Igk-eGFP bone marrow; 0.12, range: 0.09–0.13 copies per cell; SFFV-eGFP thymus; 2, range: 1–3 copies per cell; Igk-eGFP thymus; 0.10, range: 0.08–0.11 copies per cell). This suggests that the lower level of eGFP expression in Igk-eGFP mice compared to SFFV-eGFP mice may be attributed to vector dose. However, while this may explain the overall expression level differences, the reduced vector amount does not affect the specificity of the promoters as the thymi and bone marrow of Igk mice had very similar copy numbers whereas eGFP was only detected in B cells and not at all in thymi (Figure 3).
To ascertain the utility of the Igk-E promoter in human B cells, we transduced human peripheral mononuclear cells (HuPBMCs) with Igk-E-eGFP lentivirus.
Like with mouse B cells, optimum conditions for efficient lentiviral transduction of human B cells were determined by firstly culturing HuPBMCs with or without activation using CpG dinucleotides and/or Staphylococcus aureus Cowan Strain (SAC) preparations in the presence of lentiviruses containing various promoters. Total T-cell and B-cell numbers were not altered by culture conditions. Over 80% of the cells in each culture were T cells, while 10% of cells were B cells (data not shown). Culturing with CpG and CpG/SAC improved B-cell transduction efficiency compared to culturing in media alone, regardless of the promoter used. However, there were distinct differences in the relative gain in transduction efficiency by culture conditions dependent on the promoter used (Figure 4a). CpG alone resulted in the largest percentage increase in B-cell transduction for the SFFV and CMV promoters however the addition of SAC was required to improve transduction efficiency with the Igk-E promoter. T-cell transduction efficiency was not altered by the culture conditions (1–5% of T cells were transduced, data not shown). To assess the specificity of the Igk-E promoter in human cells, HuPBMCs were cultured with SAC/CpG and transduced with Igk-E-eGFP, SFFV-eGFP or CMV-eGFP lentivirus, and cells analysed by flow cytometry 3–5 days after addition of virus. Lineage-specific transduction by each of the three viral promoters was assessed for CD19+ (B cell), CD3+ (T cell) and CD19-/CD3- (NK cell) mononuclear (MNC) populations. Representative flow cytometry plots of eGFP expression by MNC lineage following CpG/SAC stimulation are shown (Figure 4b). Expression of the eGFP from the Igk-E-eGFP lentivirus was seen exclusively in B cells, with no eGFP detected in T cells or NK cells. CMV-eGFP lentivirus preferentially expressed eGFP in B cells, however 5% of the T-cell population and 11% of the NK cell population were also eGFP+. SFFV-eGFP lentivirus drove eGFP expression in B cells (75%), T cells (5%) and NK cells (32%). The numbers of positive cells from the Ig-E-eGFP construct, using identical MOI, was as high, or in some B-cell samples even higher, as compared to the levels resulting from the CMV- or SFFV-constructs. Thus, these data demonstrate that human B cells can be efficiently transduced with lentivirus and the Igk-E promoter can drive B cell-specific expression of eGFP in HuPBMCs.
We have shown that we can achieve efficient and largely B cell-specific expression of eGFP from the Igk-E promoter in a second-generation SIN lentiviral vector in vivo in mice and in primary human activated B cells. The in vivo assessment after long-term reconstitution revealed that CD19+ cells expressed good levels of eGFP, whereas neither T cells (CD3+), myeloid cells (CD11b+), dendritic cells (DC) (CD11c+) nor neutrophils (GR-1+) were significantly eGFP+. The eGFP+ B cells comprised of both marginal zone and recirculating follicular B cells. In addition, both Igk+ as well as Igλ+ B cells appeared to express eGFP at levels comparable to SFFV-eGFP controls. This shows, as expected, that the Igk/Igλ determination process in the B cells following reconstitution and B-cell development does not affect the ability of the short Igk promoter to support transcription. For reasons that we do not understand, both promoters resulted in significantly lower levels of expression in Igλ+cells as compared to in Igk+cells.
We were only able to achieve significant transduction of differentiated mouse, as well as human B cells after or concomitant with activation. Reports in the literature vary to some degree regarding the ability of different lentiviral constructs to transduce resting non-activated mouse, as well as human, B cells. However, it appears likely that some HIV-1 accessory proteins, absent from normal recombinant lentiviral preparations, are required for successful integration in at least human resting B cells.27
The availability of lentiviral vectors with B cell-specific gene expression is of great interest in experimental in vivo models since it can serve as an alternative means for expressing genes in B cells only without having to make transgenic animals. We are particularly interested in using such means to study the role of B cells in the induction of immunity to antigens as well as in tolerance to self-antigens in experimental models. B cell-specific promoters also have a potentially important future role to play in a clinical setting. The recent successful clinical gene therapy correction of X-linked severe combined immunodeficiency using γ-retroviral vectors28, 29 is likely, in the near future, to move towards the use of mainly lentiviral vector systems combined with more widespread use also for many other immune related deficiencies and diseases. In many cases it would be highly advantageous to use cell lineage-specific promoters such as those for B cells. Within the areas of transplantation and autoimmunity, several experimental studies have shown that when B cells specifically are made to present an antigen to T cells, specific tolerance to that antigen develops. Thus, expression of an offending autoantigen in an autoimmune condition in B cells only following transduction of a lentiviral construct into either bone marrow stem cells or B-lineage cells may constitute an attractive future clinical strategy. Such constructs could benefit from incorporation of the Igk-E promoter described here.
Materials and methods
Male and female C57BL/6 mice (6- to 10-week old) were purchased from Charles River (Margate, UK) and maintained in individually ventilated cages at the local animal facility, Institute of Child Health (ICH), London, UK. All manipulations were carried out in a laminar flow hood. Animal experiments were approved by the Research Ethics Committee of ICH according to Home Office Animal Welfare Legislation.
A20 B cells,30 3T3 fibroblast cells,31 293T fibroblast cells and HCQ4 CD4+ T-cell hybridomas32 were maintained in Dulbecco's modified eagles medium (DMEM) containing 4500 mg l−1 glucose and supplements (GlutaMAXTM1, Gibco, Invitrogen, Paisley, UK; 10% (v/v) fetal calf serum (FCS); 100 U ml−1 penicillin; 100 U ml−1 streptomycin). X16C8.5 B cells30 were maintained in RPMI 1640 medium containing supplements. bEnd.3 cells33 were maintained in DMEM containing 4500 mg l−1 glucose, 1.5 g l−1 sodium bicarbonate and supplements. Media for A20 and X16C8.5 B cells was further supplemented with 0.05 mM β-mercaptoethanol. All cells were maintained in a humidified incubator at 37 °C with 5% CO2.
Rat anti-mouse CD86-PE (GL1), rat anti-mouse CD19-PE (1D3), rat anti-mouse I-Aq-biotin (KH116), rat anti-mouse CD3-PE and PE-Cy5 (17A2), rat anti-mouse CD8-PE (53–6.7), rat anti-mouse CD5 APC (53–7.3), rat anti-mouse CD21-PE (7G6), rat anti-mouse C23-biotin (B3B4), rat anti-mouse IgD (11-26c.2a), rat anti-mouse CD11b-PE and PerCP-Cy5.5 (M1/70), rat anti-mouse IgM-biotin (R6-60.2), hamster anti-mouse CD11c-biotin (HL3) and streptavidin-PE and -PE-Cy5 antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Rat anti-mouse CD19 PE-Cy5, rat anti-mouse κ and λ biotin were from Serotec (Oxford, UK). Rat anti-mouse B220-PE-Cy5 (RA3-6B2), rat anti-mouse CD4-PE-Cy5 (L3T4) and rat anti-mouse Gr-1-PE (RB6-8C5) were from eBiosciences (San Diego, CA, USA). Goat anti-rat Ig-PE was from Jackson (West Grove, PA, USA). Goat anti-mouse Ig (polyclonal)-biotin antibody was from DAKO (Cambridgeshire, UK). Mouse anti human CD19-Tricolour (MHCD1906) and mouse anti-human CD3-PE (MHCD1906) were purchased from Caltag (Buckingham, UK).
Generation of constructs
The Igk-E-eGFP lentiviral vector was constructed in three parts. First, Igk-E-was created by cloning the 3′ enhancer, intron enhancer and Igk minimal promoter into pUC19 (a gift from S Petterson, Karolinska Institute, Sweden).20 3′ enhancer (1100 bp) was removed from pIC20H by StuI digestion and ligated into SmaI-digested pUC19. Murine intron enhancer (1000 bp), a gift from S Pettersson (Karolinska Institute, Sweden),21 was liberated from the pucCGAT vector by XbaI digestion and ligated into XbaI-digested pUC19-3′ enhancer. Finally, the Igk minimal promoter19 was rescued from the Kpr-LX-CAT-SV vector by HindIII/PstI digestion, blunted and ligated into HincII/PstI-digested and blunted pUC19-3′-enhancer-intron-enhancer, to create pUC19-Igk-E. eGFP was digested from pHR’SIN-cPPT-SEW using BamHI and NotI and ligated into BamHI/NotI-digested pBluescript II KS (+/−) (Stratagene, CA, USA) to create pBluescript-eGFP. Igk-E was liberated from pUC19-Igk-E by KpnI and HindIII and ligated into KpnI/HindIII-digested pBluescript-eGFP to create pBluescript-Igk-E-eGFP. Igk-E-eGFP was liberated by digestion with KpnI and NotI and ligated into KpnI/NotI-digested pENTR1A (Gateway, Invitrogen). The Igk-E-eGFP insert was then shuttled into a HIV-1-based lentivirus Gateway destination vector (based on pHR-SIN-cPPT) previously constructed in our laboratory, (S Howe, unpublished), by Gateway LR clonase reaction (Invitrogen). The final vector was called LNT-Igk-E-eGFP. pHR’SIN-cPPT-SEW has been previously described.22 It is termed LNT-SFFV-eGFP in experiments described in this paper. LNT-CMV-eGFP has been previously described.22
VSV-G-pseudotyped lentivirus was produced by transient transfection of 293T cells with three plasmids: the self inactivating transfer vector plasmid LNT-SFFV-eGFP, LNT-CMV-eGFP or LNT-Igk-E-eGFP and the multi-deleted packaging plasmids pCMVΔR8.91 and pMD.G234 that encode structural protein and the VSV-G envelope, respectively, as previously described.35 Viral vector titre of eGFP-containing preparations was determined by transducing A20 cells with serial dilutions of virus and monitoring expression after 3 days by flow cytometry.
Lentivirus transduction of murine cell lines and primary murine cells
A20, X16C8.5, 3T3-IAq or HCQ4 cells (5 × 104) were seeded per well of a 12-well plate in 1 ml cell-specific media. Cells were infected with SFFV-eGFP- or Igk-E-eGFP-expressing virus at MOI 100. Spleen suspensions were prepared as previously described36 and enriched for B cells by depletion of CD3+ and CD80+ cells by MACS separation according to the manufacturer's instructions (Miltenyi Biotech, CA, USA). The unbound, B cell-enriched fraction was collected and resuspended at 1 × 106 cells ml−1 in RPMI 1640 medium containing GlutaMAX1, supplemented with 10% FCS, 100 U ml−1 penicillin, 100 U ml−1 streptomycin and 0.05 mM β-mercaptoethanol. Cells (1 ml) were seeded per well of a 12-well plate, with or without 10 μg ml−1 LPS and SFFV-eGFP- or Igk-E-eGFP-expressing virus at a MOI of 10. When lentivirus was added, plates of cells were spun at 700 g for 1 h at 20 °C. Cells were maintained in a humidified incubator at 37 °C, 5% CO2. eGFP expression was analysed by flow cytometry 4–7 days later.
Bone marrow transduction and reconstitution
Bone marrow was harvested from the femur and tibia of male C57BL/6 mice and Lin− cells purified using StemSep Mouse Haematopoietic Progenitor Enrichment cocktail and column (0.6″ column), according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC, Canada). Recovered Lin− cells were resuspended at 1 × 106 cells per ml in StemSpan serum-free expansion medium (StemCell Technologies) containing with 1% (v/v) FCS, 100 U ml−1 penicillin and 100 U ml−1 streptomycin and 100 ng ml−1 murine stem-cell factor (mSCF), 100 ng ml−1 mFlt-3, 100 ng ml−1 hIL11 and 20 ng ml−1 mIL3. SFFV-eGFP or Igk-E-eGFP lentivirus was added to each well at an MOI of 50. Transduced cells were cultured overnight in a 24-well plate at 37 °C, 5% CO2 in a humidified incubator, then 2 × 105 cells intravenously injected into each lethally irradiated (1000 rad, split dose) female C57BL/6 mouse. Peripheral blood and lymphoid tissues were analysed 11 (experiment 1) and 16 (experiment 2) weeks post-transplant by flow cytometry for eGFP expression and lineage markers.
Determination of eGFP lentiviral copy number by PCR
Genomic DNA was purified from cell pellets by NP40 lysis followed by incubation with proteinase K for 2 h at 56 °C. The proteinase K was inactivated at 95 °C for 15 min and the supernatant containing the genomic DNA frozen at −20 °C until used.
Quantitative PCR has been previously described37 and was performed using oligonucleotide primers and taqman probes designed and used previously37 using modified methods on an ABI 7000 SDS (Applied Biosystems, Warrington, UK). Briefly 5 μl of genomic DNA was amplified with 20 μl of qPCR supermix-UDG with ROX (Invitrogen) containing 200 μM forward and reverse primers (Sigma, Poole, UK). These were specific for sequences in the WPRE to determine viral copies and either β actin for human cells or titin for murine cells where appropriate and 100 μM 6-FAM-TAMRA probe (MWG Biotech AG, Ebersberg, Germany) for 40 cycles at 95 °C (15 s), then 60 °C (1 min). Serially diluted plasmid DNA containing the relevant sequences were used as a standard curve, with all measurements performed in triplicate.
Human peripheral blood mononuclear cell expansion and transduction
HuPBMCs were isolated from peripheral blood from two donors by density gradient centrifugation, washed and resuspended in freshly prepared RPMI 1640 medium (Gibco, Invitrogen), supplemented with 10% (v/v) FCS, 25 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine and 0.05 mM. β-mercaptoethanol. To compare culture conditions for cell type and transduction efficiency, HuPBMCs (5 × 105 per ml) were cultured in 24-well plates with or without unmethylated DNA (CpG, 6 μg ml−1, 2006, Alta Biosceinces, Birmingham, UK) and/or SAC (1:10 000, 2014, Sigma) at 37 °C in 5% CO2. Lentivirus driving eGFP expression (LNT-SFFV-eGFP, LNT-CMV-eGFP, LNT-Igk-E-eGFP) was added on day 2 of culture at a MOI of 20. Cells were harvested at 5–7 days and analysed by flow cytometry.
One to three million cells were incubated with antibodies and analysed by flow cytometry on a Coulter Epics XL flow cytometer (Beckman Coulter, CA, USA) using Expo32 ADC Software (Beckman Coulter) for acquisition and Summit v4.1. Build 2141 (DAKO, CO, USA) software for analysis.
Transduction efficiency was analysed by the one and two-tailed students t-test for lentiviral specific (Figure 4b) and pooled (Figure 4a) data, respectively. A paired students t-test was used to analyse cell type-specific eGFP expression in individual mice (Figure 3c).
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We thank the Plasmid Factory (Bielefeld, Germany) for production of plasmids pCMVΔR8.91 and pMD.G2. These studies were supported by the Arthritis Research Campaign (KG), Wellcome Trust (AJT), Leukemia Research Fund LRF (WQ) and EU grant 6th Framework (CONSERT) (SH).
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Laurie, K., Blundell, M., Baxendale, H. et al. Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector. Gene Ther 14, 1623–1631 (2007). https://doi.org/10.1038/sj.gt.3303021
- immunoglobulin promoter
- B cells
- gene transfer
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