Epidermal stem cells – possible therapeutic uses

Journal of Investigative Dermatology Symposium Proceedings (2004) 9, 269–275; doi:10.1111/j.1087-0024.2004.09302.x

Lentivirus-Mediated Gene Transfer to Human Epidermis

Soosan Ghazizadeh1, Anne B Katz, Robin Harrington and Lorne B Taichman

Department of Oral Biology and Pathology, Stony Brook University, Stony Brook, New York

Correspondence: Soosan Ghazizadeh, PhD, Department of Dermatology, VC15-208, College of Physicians & Surgeons, Columbia University, New York, USA. Email: sg2163@columbia.edu

1Present address: Department of Dermatology, College of Physicians and Surgeons, Columbia University, New York, USA.

Received 2 July 2003; Revised 2 July 2003; Accepted 4 November 2003.

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Abstract

For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably transmitted to and expressed in descendant cells. Retroviral vectors are highly efficient in gene transfer to human keratinocyte stem cells in culture; however, they cannot transduce quiescent stem cells in vivo. As lentiviral vectors (LVV) transduce non-proliferating cells, their ability to target human epidermal stem cells was evaluated. LVV were highly efficient in gene transfer to clonogenic keratinocytes in vitro. Despite higher transgene DNA content and comparable levels of transgene mRNA, levels of transgene product directed by lentivectors were 3-folds lower than that of retrovectors. When transduced keratinocytes were grafted onto mice, transgene expression persisted for at least 20 wk; however, transgene product was detected primarily in the uppermost layers of epidermis. Inclusion of an element that is known to facilitate nuclear export of intron-less transcripts, resulted in enhanced transgene expression in keratinocytes. In vivo transduction of xenografted human skin with these vectors resulted in efficient gene transfer to epidermal progenitor cells. These results demonstrate stem cell transduction by LVV and point out the utility of using these vectors for direct gene transfer to and sustained expression in human epidermis.

Keywords:

gene expression, keratinocyte, persistent, stem cells

Abbreviations:

beta-gal, beta-galactosidase; CMV, cytomegalovirus early promoter; GFP, enhanced green fluorescent protein; LVV, lentiviral vectors; MOI, Multiplicity of infection; RVV, retroviral vectors; WPRE, woodchuck hepatitis post-transcriptional regulatory element; X-gal, 4-chromo-5-bromo-3-indolyl-beta-D-galactoside

Gene therapy approaches are being considered for a variety of cutaneous disorders for which the genetic basis has been identified and available therapies are inadequate (Khavari, 2000;Uitto and Pulkkinen, 2000). For long-term cutaneous gene therapy, the therapeutic gene must be targeted to long-lived stem cells, and be stably transmitted to and expressed in descendant cells. In epidermis, stem cells carry out a life-long process of regeneration and renewal. Stem cell progeny either remain as stem cells or give rise to transit amplifying cells which, after a few rounds of replication, undergo terminal differentiation and desquamate (Hall and Watt, 1989). Therefore, only gene delivery vehicles that assure stable integration into the stem cell genome are relevant. Consequently, retroviral vectors (RVV) and lentiviral vectors (LVV) are most useful for long-term gene therapy. Although both vectors integrate into the host genome, the entry of retroviral pre-integration complex into the nucleus is dependent on the breakdown of the nuclear membrane during M phase (Roe et al, 1993), and therefore transduction is limited to proliferating cells. Lentiviral genome, however, can be transported through nuclear pores and integrated into quiescent as well as proliferating cell genome (Naldini et al, 1996b).

There are two approaches to cutaneous gene transfer, the ex vivo approach in which keratinocytes are grown from skin biopsies and genetically engineered in vitro prior to grafting, and the in vivo approach that involves direct administration of genetic material to intact epidermis. Initial approaches to cutaneous gene therapy have focused on ex vivo strategies, as there is substantial experience regarding in vitro growth and expansion of epidermal keratinocytes and their clinical application for treatment of burn victims (Green, 1991;Compton et al, 1998). RVV have been successfully used for gene transfer and long-lasting transgene expression in both keratinocyte culture (Mathor et al, 1996) and transplantation onto immunodeficient mice (Kolodka et al, 1998). Ex vivo models of gene therapy for several monogenic skin disorders including epidermolysis bullosa subtypes and recessive ichthyoses have been developed (Freiberg et al, 1997;Dellambra et al, 2000;Robbins et al, 2001;Ortiz-Urda et al, 2002).

Ex vivo approaches to cutaneous gene therapy, however, are complicated by significant scarring that typically accompanies skin autografts. Moreover, the lack of epidermal appendages in these grafts undermines their capacity to provide normal skin function. Direct long-term gene transfer to epidermal stem cells would circumvent these problems. RVV have been used for direct gene transfer to mouse epidermal stem cells; however, mitotic activation of stem cells by dermabrasion was required for successful gene transfer (Ghazizadeh et al, 1999). Direct injection of RVV into human skin grafted onto immunodeficient mice has not yielded similar results, partly due to distinct differences in structure and cell kinetics between mouse and human epidermis (Gelfant, 1982;Baek et al, 2001).

Efficient in vivo gene transfer and expression by LVV have been demonstrated in several other tissues including muscle, neural, and liver tissues (Naldini et al, 1996b;Blomer et al, 1997;Kafri et al, 1997). Intradermal injection of LVV into human skin resulted in persistent gene expression in skin; however, dermal fibroblasts were the primary targets (Baek et al, 2001). A systematic evaluation of lentivirus-mediated in vivo gene transfer to epidermis demonstrated low transduction efficiency with no evidence for stem cell targeting. Furthermore, these vectors had no advantage over that of RVV in gene transfer into keratinocytes progenitor cells in vitro (Kuhn et al, 2002), despite the presence of slower cycling of keratinocyte stem cells in culture (Albers et al, 1986). The objective of this study was to examine potential mechanisms for the poor performance of LVV observed in gene transfer to epidermal stem cells. Here we demonstrate that LVV are highly efficient in gene transfer and integration to keratinocyte stem cells in vitro and in vivo; however, lentivirus-mediated transgenes are not properly expressed in epidermal keratinocytes.

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Results

LVV are highly efficient in gene transfer to clonogenic keratinocytes in culture

Cultures of human keratinocytes contain stem cells, transit amplifying cells and terminally differentiating cells (Barrandon and Green, 1987), and it is likely that stem cells in culture replicate more slowly than transit amplifying cells (Albers et al, 1986). We have previously shown that clonogenic keratinocytes capable of more than 30 cumulative population doublings behave as stem cells in vivo, that is, persist for prolonged periods and generate differentiated epidermis when grafted onto immunodeficient mice (Kolodka et al, 1998). As LVV transduce both proliferating and quiescent keratinocytes (Kuhn et al, 2002), they are likely to be more efficient than RVV in gene transfer to slowly cycling stem cells. To test this hypothesis, subconfluent cultures of normal human keratinocytes were transduced with a Maloney murine leukemia virus-based retroviral vector (MFGLZ) or human immunodeficiency virus-based lentiviral vector (HIVLZ) encoding lacZ at various multiplicity of infections (MOI) ranging from 0.5 to 8. Two days after transduction, cells were reseeded at clonal density and 2 wk later, stained for beta-gal activity using X-gal staining. As shown in Figure 1a, HIVLZ was more efficient than MFGLZ at transducing clonogenic keratinocytes at each MOI tested. These differences, however, were more pronounced at lower MOI, as there was 4-, 2-, and 1.3-fold-enhanced transduction by HIVLZ at MOI of 1, 4, and 8, respectively. The higher rates of transduction of clonogenic cells by LVV suggest that these vectors are more efficient in transduction of slow cycling stem cells.

Figure 1.
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Lentivirus-mediated transduction of clonogenic keratinocytes in culture. (A) Transduction efficiencies of clonogenic human keratinocytes transduced at multiplicity of infection (MOI) of 0.5, 1, 2, 4, 8 with HR'CMVLZ-SIN (HIVLZ) or MFGLZ were measured by X-gal staining of established colonies of keratinocytes. Error bar indicates standard deviation for each group (n=3) in three separate experiments. (B) Transduced clones were expanded for more than 30 population doublings and beta-gal activity in transduced clones (n=18) from each group was quantified by flow cytometry using CMFDG as a substrate and expressed as mean fluorescent intensity.

Full figure and legend (22K)

Although HIVLZ was more efficient in transduction of keratinocytes, levels of beta-gal enzyme activity in transduced cells were significantly lower than that of MFGLZ-transduced keratinocytes, as the time required for X-gal staining of HIVLZ-transduced keratinocytes was longer (data not shown). To quantify these differences, beta-gal activity in transduced clones was quantified by incubation of cells in media including 5-chloromethylfluorescein D-galactopyranocide (CMFDG) substrate reagent, and cellular fluorescent products were analyzed by flow cytometry. As indicated in Figure 1b, the average mean fluorescent intensity (MFI) in MFGLZ-transduced clones was approximately three to four times higher than that of HIVLZ-transduced clones, indicating significantly lower levels of transgene expression directed by LVV.

LVV-mediated transgene expression is regulated post-transcriptionally

MFGLZ and HIVLZ use different promoters to derive transgene expression. Transgene expression in MFGLZ is directed by viral promoter located in the long terminal repeat (LTR) while in HIVLZ, lac Z expression is driven by an internal cytomegalovirus (CMV) promoter. To explore whether the low level of beta-gal activity in HIVLZ-transduced keratinocytes was due to lower levels of transcription, keratinocytes were transduced with HIVLZ or MFGLZ at MOI of 10. At this MOI more than 95% of keratinocytes were transduced and expressed beta-gal. Polyclonal cultures of transduced keratinocytes were expanded and used for comparative protein, DNA and RNA analyses (Figure 2). Similar to that observed for transduced clonogenic keratinocytes, beta-gal activity in MFGLZ-transduced cultures was four times higher than that in HIVLZ-transduced cultures (Figure 2a). Southern blot analysis, however, demonstrated higher copy numbers of lacZ transgene in the HIVLZ-transduced population, indicating enhanced lentivirus transduction and integration (Figure 2b). But analysis of total RNA with a lacZ-specific riboprobe indicated comparable levels of lacZ transcript in both MFGLZ- and HIVLZ-transduced cultures. These data indicated that the differences observed in levels of beta-gal activity are not reflective of steady-state lacZ transcript levels (Figure 2c) and suggested that post-transcriptional mechanisms are involved in regulating LVV-mediated transgene expression.

Figure 2.
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Post-transcriptional control of lentiviral-mediated transgene expression in keratinocytes. Human keratinocytes were transduced with MFGLZ or HIVLZ at an MOI of 10 (95%–96% expressing lacZ). Transduced cells were expanded and used for comparative protein, DNA and RNA analyses. Non-transduced was used as a control. (A) beta-gal activity in control and transduced keratinocytes was analyzed by flow cytometry and expressed as MFI. (B) Southern analysis of genomic DNA isolated from control and transduced keratinocytes as indicated in the figure and hybridized with a lacZ-specific probe. (C) Total RNA isolated from non-transduced (lane 1), MFGLZ- (lane 2) and HIVLZ-transduced keratinocytes (lane 3) and steady-state mRNA levels for lacZ and beta-actin were measured by hybridizing 10 mug total cellular RNA with specific antisense riboprobes (a 635-nt for lacZ and 270-nt for beta-actin). RNase protected fragments were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel.

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Expression pattern of lentiviral-delivered transgene product in epidermis

To evaluate lentivirus-mediated transgene expression in epidermis, organotypic cultures were constructed from polyclonal cultures of HIV-LZ or MFG-LZ transduced keratinocytes and grafted onto nude mice. Twenty weeks later following at least five epidermal turnovers (Kolodka et al, 1998), biopsies were obtained and beta-gal expression in cryosections was assessed by X-gal staining (Figure 3). Histological examination of grafts generated from MFGLZ-transduced keratinocytes showed distinct columns of beta-gal positive cells (blue staining) extending from basal to cornified layers of epidermis (Figure 3a). In grafts generated from HIVLZ-transduced keratinocytes, however, X-gal stained cells were observed in the uppermost granular and cornified layers of epidermis (Figure 3c). No staining was detected in the lower layers of epidermis. Longer periods of incubation in X-gal solution resulted in faint staining of cell clusters in the lower layers of HIVLZ-transduced epidermis (Figure 3d, arrows), contrary to the intense blue staining of MFGLZ-transduced tissue. These results indicated persistence of transgene in the epidermis regenerated from HIVLZ-transduced keratinocytes; however, lentivirus-mediated transgene expression was suppressed in the lower layers of epidermis.

Figure 3.
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Histochemical staining of grafted tissue for beta-gal expression. Organotypic raft cultures were constructed with MFGLZ-transduced keratinocytes (A and B) or HIVLZ-transduced keratinocytes (C and D) and were grafted onto athymic mice. Twenty weeks later tissue was excised and cryosections were examined for beta-gal expression by staining with X-gal for 1 h (A and C) or 3 h (B and D). beta-gal expressing cells are stained blue. Note lacZ expression in the transitional and cornified layers of epidermis in C. Sections were counterstained with H&E. Bar represents 50 mum.

Full figure and legend (70K)

Inclusion of elements known to facilitate RNA export in the vector enhance lentivirus-mediated transgene expression

Confinement of lentivirus-mediated lacZ expression to the uppermost layers of epidermis, a point at which nuclear disintegration is known to occur (Leigh and Watt, 1994) suggests a block to export nuclear LVV transcripts. A post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) has been shown to facilitate nuclear export of intron-less transcripts (Huang and Yen, 1995) and has been shown to enhance retrovirus- and lentivirus-directed transgene expression (Zufferey et al, 1999). To test whether inclusion of WPRE in the vector results in enhancement of transgene expression in keratinocytes, this element was inserted in a sense orientation downstream of enhanced green fluorescent protein (GFP) reporter gene in pHR'cytomegalovirus (CMV)-GFP to generate pHR'CMV-GFP-WPRE. GFP was used instead of lacZ for technical simplicity and to demonstrate that transgene expression pattern in lentivirus-transduced cells is independent of transgene. Normal keratinocytes were transduced with either pHR'CMV-GFP or pHR'CMV-GFP-WPRE and GFP expression was examined by flow cytometry. As indicated in Figure 4, inclusion of WPRE in pHR'CMVGFP resulted in 3-fold increase in the levels of GFP in cultured cells. To assess GFP expression in epidermal tissue, organotypic rafts were constructed from transduced keratinocytes and analyzed histologically. Examination of cryosections indicated GFP expression in the upper layers of epidermis in pHR'CMV-GFP (Figure 4b); however, in organotypic cultures generated from pHR'CMV-GFP-WPRE, GFP was expressed in all layers of epidermis (Figure 4c). These data indicated that inclusion of WPRE element could overcome the restrictions observed in LVV-mediated transgene expression in epidermis.

Figure 4.
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WPRE enhances lentivirus-mediated transgene expression. (A) Kertinocytes were transduced with HR'CMV-GFP or HR'CMV-GFP-WPRE at an MOI of 0.3 or 3. Three days post-transduction, GFP expression in HR'CMV-GFP-transduced cells (black bars) was compared with that of HR'CMV-GFP-WPRE-transduced cells (gray bars). The percent GFP positive cells in each population is stated above the bars. This is a representative of three independent experiments. (B and C) Three-dimensional raft cultures of human keratinocytes were constructed using keratinocytes transduced with HR'CMV-GFP (B) or HR'CMV-GFP-WPRE (C) at an MOI of 3. Rafts were fixed, cryopreserved and sections were counterstained with DAPI (blue nuclear staining) and analyzed for green fluorescent (green). The position of basement membrane is indicated with a dotted line. The bar represents 50 mum.

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LVV are efficient in direct gene transfer to human progenitor cells in vivo

The potential advantage of LVV in cutaneous gene therapy is their ability to target slowly cycling epidermal stem cells in vivo. To examine the feasibility of direct gene transfer to human epidermis, human foreskins were grafted onto nude mice and were injected intradermally with concentrated pHR'CMV-GFP-WPRE (1 times 107 infectious particles in 20 muL volume). Initial GFP expression in the transduced skin was assessed at 1 wk post-transduction in live animals using fluorescent stereoscopy. As shown in Figure 5a and b, a significant number of GFP positive cells were observed in the surface of skin grafts transduced with LVV. Histological examination of transduced human skin at 10 wk post-transduction by fluorescent microscopy demonstrated GFP expression in all layers of epidermis (Figure 5c). The presence of GFP-expressing columns in epidermis at this time indicated that progenitor cells were transduced and were capable of giving rise to differentiated cell descendants. These data clearly indicate that LVV are efficient in direct gene transfer to human epidermal progenitor cells and provide a tool for long-term gene transfer and expression in epidermis.

Figure 5.
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In vivo transduction of xenograted human epidermis with LVV. Human foreskin grafts were injected intradermally with HR'CMV-GFP-WPRE (5 times 106 transducing units in 20 muL) and GFP expression was assessed in live animals using a fluorescent stereoscope (A and B). The boxed region in A is shown at higher magnification in B to show GFP-expressing corneocytes. At 12 wk post-transduction transduced skin was excised, fixed and cryosections were examined by fluorescent microscopy. Extensive GFP expression in the epidermis is shown in C. A column of GFP expressing cells representing an epidermal proliferative unit is shown in D. Tissue sections were counterstained with DAPI (blue nuclear staining). In C and D, the position of basement membrane is denoted with a dotted line. Bar represents 1.2 mm for A, 200 mum for B, and 50 mum for C and D.

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Discussion

As gene transfer vehicles, LVV's are advantageous compared with RVV because they can infect quiescent cells, and are more efficient for in vivo gene transfer (Naldini et al, 1996a). Direct injection of LVV into a variety of tissues including neural, muscle, and liver tissues as well as into quiescent human hematopoietic stem cells results in efficient gene transfer and expression (Blomer et al, 1997;Kafri et al, 1997;Uchida et al, 1998). In this study, we evaluated the ability of LVV to mediate stable gene transfer to and expression in human epidermis. Our data showed that LVV are superior to RVV in gene transfer to epidermal progenitor cells in vivo and in vitro. The higher transduction efficiency of LVV was expected, because epidermal stem cells are quiescent and not readily targeted by RVV (Bickenbach, 1981;Cotsarelis et al, 1990). Moreover, clonogenic cells in culture also cycle more slowly (Albers et al, 1986) and under-represented in the RVV-transduced cultures (Kolodka et al, 1998). The differences between efficiency of gene transfer to clonogenic cells mediated by LVV and RVV were more pronounced at low MOI, as at high virus concentration, almost all keratinocytes were transduced by either vector. The enhanced gene transfer to keratinocyte stem cells by LVV was not appreciated in studies ofKuhn et al, 2002, as cultured keratinocytes were transduced with an MOI of 25.

The higher transduction efficiency by LVV was reflected in a higher copy number of integrated transgene DNA. Interestingly, despite these higher copy numbers, steady-state transgene mRNA levels were comparable between RVV- and LVV-transduced populations. A direct comparison between transcriptional activities of the two vectors is difficult since the promoters driving lacZ expression in these vectors are not identical. Surprisingly, despite comparable levels of steady-state transgene mRNA in the two populations, levels of transgene product in LVV-transduced keratinocytes was 3–4-fold lower than that of RVV-transduced cells. This lower level was not transgene dependent as similar results obtained when lacZ was replaced with GFP. Even more surprising was the pattern of transgene expression observed in epidermis regenerated from LVV-transduced keratinocytes. High levels of transgene expression were confined to terminally differentiated keratinocytes in the upper-granular and the cornified layers of epidermis where nuclear disintegration is known to occur (Leigh and Watt, 1994). The high levels of transgene expression in upper strata suggested a block in export of lentiviral RNA from nucleus to cytoplasm in keratinocytes, as high levels of transgene expression in keratinocytes were correlated with nuclear degredation (Figure 3). We explored this correlation by incorporating the WPRE into the LVV genome and noted improved expression, which suggested that a block to nuclear export did limit LVV-transcript expression.

Our data demonstrated stable and high efficiency of gene transfer to human epidermal cells following direct intradermal injection of WPRE-containing LVV. Transgene expression persisted at least for 12 wk (duration of analysis) corresponding to three to four epidermal regeneration in human epidermis (Potten, 1981). Expression of transgene in epidermis was confined to vertical columns representing epidermal proliferating units (Potten, 1974). This is the first report demonstrating an extensive and durable transgene expression following direct gene transfer to human epidermis. The previous attempts in direct LVV-mediated gene transfer either resulted in no gene expression by 10 d post-transduction (Kuhn et al, 2002), or low efficiency of epidermal gene transfer (Baek et al, 2001). The extensive epidermal gene expression in our studies likely reflects the use of improved LVV as the number of particles injected was similar to that used in other studies.

In summary, our data indicated that LVV are highly efficient in gene transfer to epidermal stem cells. Unlike previous studies with in vivo transduction of mouse skin (Ghazizadeh et al, 1999), LVV-mediated transduction of human epidermis involves little trauma or inflammation. This improvement in direct gene transfer and expression in human epidermis provides a powerful research tool and helps to bring the promise of long-term cutaneous gene therapy one step closer to reality.

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Material and Methods

Cell cultures and construction of organotypic cultures

Human foreskin keratinocytes were grown in submerged cultures in the presence of irradiated 3T3 cells (Rheinwald and Green, 1975) using keratinocyte medium described byWu et al (1982). 293T were grown in Dulbecco's minimal essential medium (Gibco/BRL, Grand Island, New York) supplemented with 10% fetal bovine serum.

Construction of the organotypic cultures has been described previously (Kolodka et al, 1998). Briefly, transduced keratinocytes were seeded onto the collagen matrix containing human dermal fibroblasts, submerged in the media and maintained for 4 d before raising to the air–liquid interface for an additional 8 d. Cultures were either grafted onto Swiss nu/nu mice (Taconic Farm, New York) as described previously (Kolodka et al, 1998), or processed for histology.

Viral vectors and transduction

Vesicular somatitis virus (VSV)-G-pseudotyped RVV encoding lacZ (MFGLZ) or enhanced green fluorescent protein (LZRS-GFP) as reporter genes have been described previously (Ghazizadeh et al, 1999;Ghazizadeh et al, 2002). The expression of transgene in these vectors is driven by viral promoter in LTR. Self-inactivating LVV that express lacZ or GFP driven by an internal CMV promoter including pHR'CMV-LZ, pHR'CMV-GFP, and pHR'EF1alpha-GFP-WPRE were kindly provided by Dr Didier Trono (CMU, Switzerland). The WPRE from the latter vector was used to construct pHR'CMV-GFP-WPRE. Lentiviruses were generated by transient cotransfection of three plasmids into 293T cells including the packaging construct pCMVDelta8.9, an envelope coding plasmid (pCMV-VSV-G), and the transgene construct. The virus-containing supernatant was collected 36–72 h after transfection, filtered through 0.45 mum filter (Gelman Science, Ann Arbor, Michigan), and stored at -70°C. For in vivo transduction, filtered viral supernatant was concentrated 1000-fold by ultracentrifugation as described elsewhere (Burns et al, 1993). Viral titers were 1–3.0 times 106 transducing unit per mL as determined on 293T cells, for all viral stocks used for in vitro transduction and 5 times 108 transducing units per mL for those used for in vivo transduction.

In vivo transduction

Direct gene transfer to mouse epidermis has been described (Ghazizadeh et al, 1999). As a model for direct gene transfer to human epidermis, neonatal foreskin was grafted onto a full thickness, circular 12 mm wound that was created on the dorsum of 6-wk-old NIH male Swiss nu/nu mice. Grafts were covered with Vaseline gauze and a polyvinyl bandage to prevent scratching and facilitate tissue engraftment. The dressing was removed after 10–14 d. Six weeks post-grafting, 20 muL (containing 1 times 107 CFU) of pHR'CMV-GFP-WPRE were injected intradermally into the grafted skin with the aid of a 30 gauge needle. At 10 wk post-transduction, transduced skin was excised, fixed for 30 min in 4% paraformaldehyde on ice, washed in phosphate-buffered saline (PBS), soaked for 30 min in 30% sucrose and cryopreserved. Animal studies were performed in accordance with institutional guidelines set forth by the State University of New York.

Detection of transgene products

For detection of beta-gal expressing cells, submerged cultures or cryosections obtained from transduced tissue were fixed for 10 min in 0.2% gluteraldehyde and stained en face with 1 mg per mL X-gal in 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mM MgCl2, 0.02% NP-40 and 0.01% sodium deoxycholate until the blue staining developed. DetectaGene green CMFDG lacZ gene expression kit (Molecular Probe, Eugene, Oregon) and flow cytometry were used in some experiments to quantify beta-gal activity in transduced keratinocytes.

For detection of GFP, transduced cells grown in submerged cultures were trypsinized, fixed in 2% paraformaldehyde and analyzed for GFP expression by flow cytometry on a Becton Dickinson FACScan (BD Biosciences, San Jose, California). Skin-directed GFP expression in live animals was assessed using a fluorescent stereoscope model Bio2-M (Zeiss, Thornwood, New York) equipped with a mercury 100 W lamp power supply and a proper filter set for GFP detection. For detection of GFP in transduced skin, cryosections were fixed for 10 min in 4% paraformaldehyde, rinsed in PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and examined by fluorescent microscopy.

DNA and RNA analyses

For Southern blot analysis, 5 mug of genomic DNA extracted from transduced cells was digested with BglI, electrophoresed and transferred to nylon membrane. Equal gel loading was verified by ethidium bromide staining. A 689-bp digoxigenin-labeled DNA probe specific to lacZ was used for hybridization. The hybridized probe was detected using the DIG/Genius Labeling Kit (Roche, Indianapolis, Indiana) according to the manufacturer's recommendations. Steady-state lacZ transcript levels were quantified by RNase protection assay using a 635-nt antisense riboprobe specific to lacZ as described previously (Ghazizadeh et al, 1997). A 270 nucleotide for beta-actin was used as internal control for gel loading.

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Acknowledgements

We are grateful to Ning Lin for technical assistance. This research was supported by grants from the NIH to LT (R01-DE04511) and to SG (K01-AR02100).

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