Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability

Gene 283 (2002) 199–208 www.elsevier.com/locate/gene

Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability Stefano Indraccolo a,b,*, Valeria Roni b, Rita Zamarchi b, Francesca Roccaforte b, Sonia Minuzzo b, Laura Stievano b, Walter Habeler b, Novella Marcato b, Veronica Tisato b, Valeria Tosello b, Luigi Chieco-Bianchi b, Alberto Amadori b b

a IST-Viral and Molecular Oncology Section, Via Gattamelata, 64 – 35128 Padua, Italy Department of Oncology and Surgical Sciences, University of Padua, Via Gattamelata, 64 – 35128 Padua, Italy

Received 27 June 2001; received in revised form 29 October 2001; accepted 22 November 2001 Received by J. Svoboda

Abstract The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene. These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Enhanced green fluorescent protein; Endothelial cells; Enhancer; Lymphocytes; Tie2; CD2

1. Introduction Retroviral vectors derived from Mo-MLV are widely used to transfer genes of interest into a variety of animal and human cells, both in vitro and in vivo (Verma and Somia, 1997). In addition to their exploitation for gene therapy approaches, they are an useful tool for gene expression studies, because they integrate the transgene in the infected cell, and permit to analyse the activity of cis-acting Abbreviations: cfu, colony forming units; CMV, cytomegalovirus; DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; G418, Geneticin; GFP, green fluorescent protein; h, hours; HUVEC, human umbilical vein endothelial cells; LTR, long terminal repeat; MFI, mean fluorescence intensity; moi, multiplicity of infection; Mo-MLV, Moloney murine leukemia virus; neoR, neomycin-resistance gene; PCR, polymerase chain reaction; SIV, simian immunodeficiency virus; VSV-G, vesicular stomatitis virus G protein * Corresponding author. Tel.: 139-49-821-5875; fax: 139-49-807-2854. E-mail address: [email protected] (S. Indraccolo).

regulatory sequences in the context of chromosomal DNA. However, it has been shown that the inclusion of cell typespecific elements in the vector as internal promoters or enhancers does not always allow to detect their activity, due to a number of drawbacks which include promoter interference, loss of specificity and instability of the vector (Bauer et al., 1994; Vile et al., 1995; De Angioletti et al., 2001). Engineering the viral LTR with cell type-specific regulatory sequences is one of the favourite alternative approaches to transcriptional targeting, in view of the fact that the LTR drives the expression of at least one of the vector-encoded genes in most retroviral vectors. Indeed, previous studies have clearly shown that targeted transcription can be consistently achieved in Mo-MLV-based retroviral vectors when a cell type-specific enhancer is inserted in the U3 part of the viral LTR lacking the viral enhancer (reviewed in Miller and Whelan, 1997). GFP has recently been widely used as a tool to address several biological issues (Prasher, 1995; Tsien, 1998).

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00857-5

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Numerous studies demonstrate its utility to measure gene expression in vitro and in vivo, to study intracellular protein traffic, and to label unicellular organisms and specific cells in multicellular organisms (Chalfie et al., 1994; Plautz et al., 1996; Cormack, 1998; Ludin and Matus, 1998; Rizzuto et al., 1998; Kallal and Benovic, 2000). A variant of GFP with increased fluorescence intensity, termed EGFP, has been generated by site-directed mutagenesis (Yang et al., 1998), and used as reporter gene to determine gene transfer efficiency by viral vectors, including retroviral, lentiviral and adenoviral vectors (Klein et al., 1997; Grignani et al., 1998; He et al., 1998; Zufferey et al., 1999). In the past few years, we have been working with EGFP as a reporter gene to initially study gene transfer efficiency by different MLV/ SIV pseudotypes, and subsequently the activity of cis-acting regulatory sequences from the CD4 gene in Mo-MLV-based retroviral vectors (Indraccolo et al., 1998, 2000). One of the advantages of the EGFP marker is its brightness, which allows detection of transduced cells expressing even single copies of the transgene (Klein et al., 1997). This is a rather common situation following virus-mediated gene transfer, as opposed to physical transfection procedures, which usually deliver many copies of the transgene to the transfected cell. Another feature of EGFP which contributes to its wide exploitation in the field of gene transfer is the high stability of the protein in the cells, whose half-life is estimated to be greater than 24 h (Li et al., 1998). On the other hand, accumulation of EGFP in the cells can be a factor that might limit those studies aimed at addressing the activity of regulatory sequences, such as promoters and enhancers, in genetically modified cells. To facilitate these studies, a variant of EGFP has been recently developed, and termed d2EGFP (Li et al., 1998). This protein is characterized by a fusion of the N-terminus of EGFP to a destabilizing Cterminus of mouse ornithine decarboxylase which targets the protein for degradation. As a result, the half-life of d2EGFP is only 2 h in mammalian cells. It is currently not known if d2EGFP might be useful for gene expression studies exploiting retroviral vectors for gene delivery. To investigate this, we established a vector system that might be generally used to investigate the activity of cell type-specific regulatory sequences in retroviral vectors exploiting EGFP or d2EGFP as the reporter gene. We tested this system with the CD2 or the Tie2 enhancers and show that recombinant vectors containing these regulatory elements generate T lymphoid-specific and endothelial cell-specific gene expression, respectively, in established cell lines and primary cells. However, detection of enhancer activity only occurs with EGFP as the reporter gene in this system.

2. Materials and methods 2.1. Plasmids The LESN vector, a derivative of the LXSN retroviral

vector (Miller and Rosman, 1989) carrying the gene for EGFP driven by the Mo-MLV LTR, as well as a neoR gene (Fig. 1), was used as the basic vector in this study (Klein et al., 1997). To investigate promoter activity in the absence of the viral enhancer, we generated a modified LESN retroviral vector carrying a deletion in the U3 region of the LTR, and termed it LESND. This vector was generated by replacement of a 1.56 kb-long XbaI/XhoI restriction fragment from the LESN vector with a 1.37 kb-long XbaI/ XhoI-digested PCR product obtained by amplification of the LESN template with primers LXSN-for (5 0 -GTTAACTCGAGGATCCGCTGTG-3 0 ) and LXSN-rev (5 0 -GCTCTAGACCTTGATCTGAACTTCTCTATTC-3 0 ), binding to positions 1620–1641 and 2976–2991, respectively, of the LXSN genome (Miller and Rosman, 1989). Therefore, the LESND vector lacks the MLV enhancer, located between bases 2976 and 3186 of the parental LXSN genome (Srinivasan et al., 1984). To generate a retroviral vector carrying a T lymphoidspecific enhancer, a 1 kb-long sequence, containing a T cellspecific transcriptional enhancer from the human CD2 gene (Lake et al., 1990), was amplified from the VA hCD2 plasmid containing the human CD2 locus (a kind gift from D. Kioussis, MRC, London, UK) (Zhumabekov et al., 1995), by PCR using the following primers, carrying a XbaI site in the extension (Fig. 1B): LCR-for2: 5 0 -GCTCTAGA-TGACTAGACCCGTGTCTGCT-3 0 LCR-rev2: 5 0 -GCTCTAGA-GGCTGGTCTCAAACTCCT-3 0 . PCR was performed in 50 ml standard buffer containing 0.2 mM of each primer and 1 U of Taq polymerase (Perkin Elmer, Foster City, CA, USA), under the following conditions: 948C denaturation 1 min, 658C annealing 30 s, 728C extension 2 min, for 30 cycles, with 5 ng plasmid DNA as template. The PCR products were electrophoresed on 1% agarose gel, purified by the GFX kit (Amersham-Pharmacia, Uppsala, Sweden), and cloned in the pCR2.1-TOPO vector (Invitrogen, Groningen, The Netherlands) following the manufacturer’s instructions. Subsequently, inserts were excised from this vector by digestion with XbaI, and sticky-ends cloned by standard procedures in the corresponding sites of LESND. The resulting retroviral vector was termed LESND-CD2 (Fig. 1B). To generate the LESND-Tie2 vector, a 303 bp-long sequence, containing the Tie2 endothelial cell-specific transcriptional enhancer (Schlaeger et al., 1997; Fadel et al., 1999), was amplified from cDNA retrotranscribed from HUVEC by PCR using the following primers, carrying a XbaI site in the extension, and the PCR conditions described above: Tie2-for2: 5 0 -CCATGGGGACATGGCTGTCAT-3 0 Tie2-rev2: 5 0 -GCTCTAGAGGAAAGTGGCTGCT-3 0 .

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Fig. 1. Schematic representation of the rearranged Mo-MLV LTR with the deletion of the viral enhancer (panel A) and of the EGFP and d2EGFP transfer vectors containing different CD2 and Tie2 gene regulatory elements (panel B). The Mo-MLV packaging signal is present in each vector genomic-RNA encoding construct; the constructs carry a long encapsidation signal (c 1), which also comprise some gag sequences that increase packaging efficiency (Miller and Rosman, 1989). SV40, simian virus 40 promoter; neoR, neomycin phosphotransferase gene; CD2, human CD2 enhancer; Tie2, murine Tie2 enhancer. The grey box indicates the deletion of the Mo-MLV enhancer; the approximate position of the viral promoter in the vectors is indicated by the TATA-box. Restriction sites relevant for cloning are indicated: B, BamHI; X, XbaI; and Xh, XhoI. In the U3-Denh sequence insert, the position of the XbaI restriction site is underlined; the CAT and TATA box of the MLV promoter are indicated in bold.

The purified PCR fragment was first cloned in the pCR 2.1TOPO vector (Invitrogen). The purified plasmid was digested by XbaI releasing an insert of 357 bp which was cloned in the LESND retroviral vector at the XbaI site following the same protocol described for cloning of the CD2 enhancer (Fig. 1B). All constructs were verified by sequencing before performing functional assays. A 914bplong fragment containing the d2EGFP open reading frame was obtained from BamHI digestion of the construct (Clontech, Palo Alto, CA, USA), and used to replace EGFP in the different vectors described in this study. The Mo-MLV Gag-Pol expression construct gag-polgpt, which harbors the Mo-MLV gag and pol genes under the control of the Mo-MLV LTR, and a SV40 polyadenylation signal (Markowitz et al., 1988), was used to generate retroviral vector particles. This construct lacks c packaging sequences as a consequence of a 134-base-pair deletion between the Mo-MLV LTR and gag gene. A plasmid, named HCMV-G, expressing the envelope protein G of VSV under the transcriptional control of the CMV promoter, was used to pseudotype MLV particles (Yee et al., 1994).

before transfection, 1.5 £ 10 6 293T cells were seeded in 25 cm 2 tissue culture flasks. The cultures were transfected with plasmid DNA using a calcium phosphate precipitation technique (Sambrook et al., 1989). 293T and murine fibroblastic NIH-3T3 cell lines were grown in DMEM supplemented with 10% FCS (Life Technologies, Gaithersburg, MD, USA) and 1% l-glutamine. Human CD4 1CD8 1 JM cells were grown in RPMI 1640 supplemented with 10% FCS and 1% l-glutamine. Finally, HUVEC were isolated from freshly obtained umbilical cord using collagenase digestion as described (Bevilacqua et al., 1985). Primary cultures were serially passaged (,1:3 split ratio) in flasks coated with Collagen type I (Sigma-Aldrich, St. Louis, MO, USA; 10 mg/cm 2) and maintained in Medium 199 (Biowhittaker, Walkersville, MD, USA) supplemented with 20% FCS (Life Technologies), recombinant human b endothelial cell growth factor (Sigma-Aldrich 0.1 ng/ml), recombinant human fibroblast growth factor-basic (Peprotech, Rocky Hill, NJ, USA; 10 ng/ml) and heparin (50 mg/ml).

2.2. Cell culture and transfections

Infectious particles were generated by transfection of 293T cells with 3 mg of the Mo-MLV Gag-Pol expression construct pgag-polgpt, along with 6 mg of either LESN, LESND, LESND-CD2, LESND-Tie2, or the corresponding

293T human kidney cells were obtained by ATCC and used as a packaging cell line (Pear et al., 1993). The day

2.3. Transduction of cells with retroviral vectors

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vectors carrying the d2EGFP gene, as transducing vectors, and 0.3 mg of the VSV-G expression construct. Fresh medium was added to the cultures 12–18 h before the supernatant was collected and passaged though 0.45 mm-pore size filters. To determine viral titer, serial dilutions of the filtered supernatants were layered over NIH-3T3 target cells, that had been seeded into 6-well culture plates the day before infection at 6.0 £ 10 4 cells per well. Protamine sulphate (8 mg/ml) (Sigma) was added to the wells, and the cells were kept in a total volume of 2 ml. After 6–9 h at 378C, 3 ml of medium were added to dilute the protamine sulphate; 36 h later, the cells were split 1:10 in 10 cm diameter Petri dishes, and new medium containing G418 (Life Technologies, 500 mg/ml active compound) was added. Following 3-week culture in G418 selective medium, the viral titer was determined as described (Ausubel, 1988), and expressed as cfu/ml of supernatant. Transduction of NIH-3T3, JM cells and of HUVEC was performed by using cell-free supernatant. To this end, volumes of supernatants containing identical amounts of viral particles were incubated with 2 £ 10 5 target cells with moi ¼ 0.2 for 6–9 h at 378C in the presence of protamine sulfate (8 mg/ml). Lymphoid cells were then pelleted, and resuspended in fresh RPMI 1640 medium. HUVEC were transduced at passage number P6; following incubation for 6–9 h at 378C with retroviral vector-containing supernatant, in the case of both HUVEC and NIH-3T3 cells, 3 ml of complete medium were added to dilute the protamine sulphate. Later (36 h), HUVEC and NIH3T3 cells were split 1:10 in 10 cm diameter Petri dishes. Transduced cells were subsequently selected in G418-containing medium for 3 weeks prior to assessment of EGFP expression. In a set of experiments, transduced NIH-3T3 cells were analyzed for EGFP expression 72 h after transduction. 2.4. Cytofluorimetric analysis EGFP-expressing vector-transduced lymphoid cells were analyzed on an EPICS-Elite cytofluorimeter (Coulter, Fullerton, CA, USA). At different times after infection, cells were pelleted, washed, and fixed with PBS-1%formaldehyde. As a negative control, a mock-transduced cell line was analyzed in parallel to set up the cytofluorometer; the mock autofluorescence did not exceed in every case the 2%. The MFI was calculated by the following formula: MFI ¼ log10(mean £ 10) £ (1024/4). 2.5. Estimation of proviral DNA content by real-time PCR analysis Genomic DNA was extracted from aliquots of 5 £ 10 6 G418-selected transduced cells by the Easy DNA kit (Invitrogen), and diluted to 100 ng/ml in water. The EGFP copy number per 5 ml genomic DNA was estimated in duplicate using the EGFP234p real-time PCR assay (Klein et al., 2000). Amplification was performed in an ABI Prism 7700 Sequence Detector System (Perkin-Elmer). The actual

genomic DNA content of each sample and its amplificability were estimated by a second real-time PCR assay targeting the rDNA genes. The estimated proviral DNA content of each sample was standardized to the value of this second PCR assay. A positive and a negative control, represented by genomic DNA from a cell line with a known amount of proviral DNA, and the parental non-transduced cells, respectively, were used to calibrate the assay.

3. Results and discussion 3.1. Construction of retroviral vectors carrying a deletion in the viral enhancer and EGFP or d2EGFP as reporter genes To establish a system that might be useful to study cell type-specific enhancer activity in cells transduced by retroviral vectors carrying GFP variants, we generated a set of vectors carrying either EGFP or d2EGFP as reporter genes, and a modified 3 0 LTR with a deletion of the viral enhancer, and termed them LESND and Ld2ESND, respectively (Fig. 1A). Gene expression profiles in transduced cells were compared to those obtained following transduction by the parental vectors, carrying full-length LTR, termed LESN and Ld2ESN, respectively (Fig. 1A). In preliminary experiments, retroviral vector-containing supernatants were generated by transient transfection of 293T cells, titrated and used to transduce murine NIH-3T3 cells. Titration of the vectors based on the cfu assay indicated that the different vector preparations had comparable titers (Table 1), irrespective of whether they carried the EGFP or the d2EGFP genes, thus indicating that the d2EGFP gene does not impair vector production. EGFP expression in the transduced cells was detected by cytofluorimetric analysis either 72 h after gene transfer, or 3 weeks later, following G418 selection of the transduced cells. Following observation of the transduced cells under a UV microscope, the brightness of EGFP expression was much reduced in cells transduced by vectors carrying the d2EGFP marker, compared to cells expressing EGFP, Table 1 Vector titer in NIH-3T3 cells by the colony forming unit (cfu) assay a Vector

Titer (cfu/ml)

LESN LESND LESND-CD2 LESND-Tie2 Ld2ESN Ld2ESND Ld2ESND-CD2 Ld2ESND-Tie2

Mean ^ SD 4.13 ^ 0.21 £ 10 5 2.39 ^ 0.73 £ 10 5 5.01 ^ 0.12 £ 10 4 2.72 ^ 1.53 £ 10 4 2.93 ^ 0.93 £ 10 5 2.24 ^ 0.73 £ 10 5 4.26 ^ 1.75 £ 10 4 3.10 ^ 0.28 £ 10 4

a Murine fibroblastic NIH-3T3 cells were transduced with either EGFPor d2EGFP-carrying retroviral vectors and selected for 3 weeks in G418containing medium (500 mg/ml). At that time, the number of colonies was counted following staining with 2% methylene blue and the titer was determined.

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due to reduced accumulation of d2EGFP (data not shown). At FACS analysis, 72 h after gene transfer, the LESN and Ld2ESN vectors yielded higher values of MFI in transduced cells, compared to their counterparts carrying a deletion of the viral enhancer, and termed LESND and Ld2ESND, respectively (Fig. 2A). The percentage of EGFP 1 NIH-3T3 cells following transduction with the Ld2ESND vector was also reduced, compared to figures obtained with the other vectors. The presence of EGFP expressing cells following transduction by the retroviral vectors carrying the deletion of the Mo-MLV enhancer was observed in different cell types (see below) and can likely be attributed to the activity of the residual Mo-MLV promoter in the modified LTR (Fig. 1A); other publications have shown this low-level expression can be eliminated by more extensive LTR engineering – deletion of almost the complete U3 region – which might particularly be needed to address the activity of heterologous promoters (Mrochen et al., 1997; Saller et al., 1998). Moreover, a strong reduction in the MFI value was noticed in cells transduced with the d2EGFP-carrying retroviral vector carrying wildtype LTR, compared to figures obtained following transduction with the corresponding EGFP-carrying vector (Fig. 2A, panels Ld2ESN and LESN). These findings were also confirmed by analyzing EGFP expression following G418 selection, where all of the selected cells by definition carry the transgene. As shown in Fig. 2B, figures generated by the d2EGFP vectors remained much lower, in terms of MFI, than those of the EGFP-carrying viral counterparts. In addition, the percentage of Ld2ESND-transduced cells which expressed the fluorescent marker above background levels was dramatically decreased compared to figures generated by LESNDtransduced cells. A low percentage of EGFP 1 cells following transduction with the Ld2ESND vector and G418 selection was also observed with other cell types (see below), and could be due to the potential interference between the LTR and the internal SV40 promoter (Emerman and Temin, 1986), which may impair the activity of weak LTRs in particular. Overall, this experiment indicated that (I) it is feasible to generate Mo-MLV-based retroviral vectors carrying d2EGFP as a reporter gene without significant loss of titer; and that (II) the d2EGFP marker allows detection of gene transfer into NIH-3T3 cells when driven by a strong viral promoter/enhancer complex, albeit at fluorescence levels lower than EGFP. 3.2. Analysis of proviral DNA in transduced cells by realtime PCR To establish whether the observed differences in the pattern of EGFP expression in cells transduced by the different recombinant vectors might relate to differences in proviral copy number, we analyzed transduced NIH-3T3 cells by means of a quantitative PCR approach (Klein et al., 2000). This assay allows quantification of the EGFP

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gene in the transduced cell population; a multiplex realtime PCR is used for simultaneous measurement of the EGFP copy number and the cell number in a one-tube assay. The precision and accuracy of this assay compared to commonly used titration methods has been recently defined (Klein et al., 2000). Following DNA analysis by this assay, we found that NIH-3T3 cells transduced by the LESN, Ld2ESN, LESND, and Ld2ESND, and selected for the neomycin resistance, carried comparable amounts of proviral DNA (Table 2). Therefore we conclude that the different patterns of EGFP expression detected following gene delivery by the vectors under study were not associated to marked differences in the proviral DNA load in the target cells. 3.3. Activity of the lymphoid-specific CD2 enhancer in retroviral vectors To investigate whether this vector system might be exploited to address the activity of cell type-specific cellular enhancers in retroviral vectors, we generated constructs carrying a T lymphoid-specific enhancer, derived from the human CD2 gene (Lake et al., 1990), and either EGFP or d2EGFP, and termed them LESND-CD2 and Ld2ESNDCD2, respectively (Fig. 1B). Vector stocks were produced and titrated by the cfu assay; a five-fold decrease in viral titer was observed compared to the parental LESND and Ld2ESND vectors, independently from the presence of EGFP or d2EGFP in the vector (Table 1). A similar drop in titer was also found with vectors carrying the endothelial cell-specific enhancer Tie2 (see Section 3.4) and might be due to partial interference of the enhancer with the internal SV40 promoter activity, as previously shown by others with retroviral vectors carrying an internal promoter (Hantzopoulos et al., 1989; Nakajima et al., 1993). We next used these vectors to transduce both the human T lymphoid JM cell line and murine fibroblastic NIH-3T3 cells, and analyzed reporter gene expression following 3 week selection of the transduced cells in G418-containing medium. As shown in Fig. 3, this experiment disclosed that the CD2 enhancer was active specifically in JM cells, as a shift in the MFI of EGFP expression was detected at FACS analysis of LESND-CD2transduced JM cells, compared to values generated by JM cells transduced with the parental LESND vector. This increase was not associated to significant differences in the proviral DNA load among the samples, as determined by real-time PCR analysis (Table 2). As expected, EGFP expression was higher in LESN- than in LESND-CD2-transduced JM cells, in terms of MFI; this is likely due to the fact that EGFP is driven by the strong Mo-MLV enhancer in the LESN vector. On the other hand, the activity of the CD2 enhancer was not detected following transduction of JM cells with the vectors carrying d2EGFP as reporter gene; no increase in the MFI values in cells transduced by the Ld2ESND-CD2 vector compared to cells transduced with the parental Ld2ESND vector was observed (Fig. 3).

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Fig. 2. Transduction of murine fibroblastic NIH-3T3 cells by different retroviral vectors carrying EGFP or d2EGFP as a marker gene. NIH-3T3 cells were infected with either LESN, LESND, Ld2ESN, Ld2ESND retroviral vectors generated by transient transfection of 293T cells. 72 h after transduction (panel A) or 3 weeks after G418-selection (panel B), the percentage of EGFP-expressing cells was quantified by cytofluorimetric analysis. The mean percentage of EGFP-positive cells for each construct and the MFI are indicated. The profile of the mock-transduced NIH-3T3 cells is shown in each panel as a black area.

S. Indraccolo et al. / Gene 283 (2002) 199–208 Table 2 Proviral DNA content in transduced cells by real-time PCR assay a Cell type/vector

Proviral load (EGFP/cell)

NIH-3T3/LESN NIH-3T3/LESND NIH-3T3/Ld2ESN NIH-3T3/Ld2ESND JM/LESN JM/LESND JM/LESND-CD2 HUVEC/LESN HUVEC/LESND HUVEC/LESND-Tie2

1.008 1.493 1.459 1.572 1.012 1.070 1.003 0.977 0.952 1.050

a The real-time PCR-based assay was performed on genomic DNA extracted from transduced cells after G418 selection. The EGFP copy number per cell was estimated in duplicate using the EGFP234p realtime PCR assay (Klein et al., 2000). The average values reported refer to one representative experiment, which was independently repeated with comparable results.

However, JM cells transduced with the parental Ld2ESN vector showed increased expression of the reporter gene, compared to cells transduced with the vector lacking the viral enhancer, thus indicating that the activity of the strong

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Mo-MLV enhancer in JM cells can also be detected by exploiting d2EGFP as the reporter gene (Fig. 3). 3.4. Activity of the endothelial cell-specific Tie2 enhancer in retroviral vectors To extend these observations to a different enhancer, we generated retroviral vectors carrying the endothelial-specific Tie2 enhancer (Schlaeger et al., 1997; Fadel et al., 1999) (Fig. 1B), and used them to transduce HUVEC. As shown in Fig. 4A, this experiment disclosed that the Tie2 enhancer was active in HUVEC, as it induced a shift in the expression levels of the EGFP marker, compared to the parental LESND vector, in cell populations carrying comparable amounts of proviral DNA (Table 2). The MFI in HUVEC transduced by the LESND-Tie2 vector (MFI ¼ 630) was at intermediate levels between those generated by LESNDand LESN-transduced cells (MFI ¼ 490 and 763, respectively) (Fig. 4A), where the expression of the fluorescent marker is driven by the Mo-MLV enhancer/promoter complex. As found with the lymphoid-specific enhancer, a much lower increase in d2EGFP expression compared to the control vector Ld2ESND was detected following transduc-

Fig. 3. Transduction of human lymphoid JM cells by different retroviral vectors carrying EGFP or d2EGFP as a marker gene. JM cells were infected with either LESN, LESND, LESND-CD2, or the corresponding vectors carrying d2EGFP, generated by transient transfection of 293T cells, and selected for 3 weeks on G418-containing medium prior to assessment of EGFP expression by FACS analysis; the expression profile is shown by the grey line. The mean percentage of EGFP-positive cells for each construct and the MFI are indicated. The profile of the mock-transduced lymphoid cells in each panel is shown as a black line.

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Fig. 4. Cell type specificity of EGFP expression in cells transduced by retroviral vectors carrying heterologous enhancers. Endothelial (HUVEC, panel A), lymphoid (JM, panel B), and fibroblastic (NIH-3T3, panel C) cells were infected with LESN, LESND, LESND-Tie2, and LESND-CD2 vectors generated by transient transfection of 293T cells; after selection for 3 weeks in G418-containing medium, the cells were analyzed by FACS; the EGFP 1 fraction is indicated by the solid line. The mean percentage of EGFP-positive cells for each construct and the MFI are indicated. The profile of the mock-transduced lymphoid cells in each panel is shown as a black area.

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tion of HUVEC with the corresponding Mo-MLV vectors carrying the d2EGFP as the reporter gene (data not shown).

Piovan for precious help in the preparation of the manuscript.

3.5. Cell-type specificity of EGFP expression by retroviral vectors carrying heterologous enhancers

References

Finally, to investigate the cell type-specificity of EGFP expression we transduced HUVEC and fibroblastic NIH3T3 cells with the retroviral vector carrying the CD2 enhancer; moreover, JM and NIH-3T3 cells were transduced with the vector carrying the Tie2 enhancer. Results of a representative experiment are reported in Fig. 4. The lymphoidspecific CD2 enhancer was weakly active in endothelial cells (Fig. 4A) and only slightly increased EGFP expression in HUVEC; in contrast, the endothelial cell-specific Tie2 enhancer induced a much stronger MFI increase in these cells (Fig. 4A). Conversely, the Tie2 enhancer was weakly active in JM T lymphoid cells (Fig. 4B), where the CD2 enhancer showed higher activity. Concerning NIH-3T3 cells, we found that the CD2 enhancer was minimally active in these cells, as it increased the MFI only by 17 channels, compared to the enhancer-less vector LESND; the Tie2 enhancer however, was also moderately active in these cells, albeit less than in endothelial cells (Fig. 4C). Similar findings were obtained in two subsequent confirmatory experiments (data not shown), and allowed us to conclude that the CD2 and the Tie2 enhancers were preferentially active in specific cell types. 3.6. Conclusions Overall, these findings indicate that (I) it is feasible to replace the Mo-MLV enhancer with cellular regulatory elements in retroviral vectors carrying the EGFP reporter gene without significant loss of titer; (II) the CD2 and the Tie2 enhancers retain their cell type-preferential activity also in the context of a retroviral vector; (III) the vectors used transferred comparable amounts of proviral DNA in the target cells, although they carried different reporter genes (EGFP/d2EGFP) or regulatory elements; (IV) it is feasible to use d2EGFP as a reporter gene to study the activity of strong viral enhancers, such as that of MoMLV, in this system; and (V) d2EGFP might be less sensitive than EGFP to investigate the activity of cellular regulatory sequences in retroviral vectors. Acknowledgements This study was supported in part by grants from Telethon (Grant A-126); ISS (AIDS Project); MURST 60 and 40%, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Italiana per la Ricerca sul Cancro (FIRC); National Research Council (PF Biotechnology and Bioinstrumentation). S. Minuzzo is a recipient of a fellowship from FIRC; V. Roni is a recipient of an AIRC fellowship. We are also grateful to P. Gallo for artwork, and Dr. E.

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