Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter

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Abstract

Cultured epithelial grafts have proven to be life-saving in the treatment of large skin losses. It has become apparent that one of the main difficulties of this technology is the overall poor take of the grafts as a consequence of severely damaged dermal beds. Skin substitutes providing both cultured keratinocytes, as an epidermal layer, and a dermal analogous offer a more suitable material for skin repair. Ex vivo transfer of stroma regeneration-promoting genes to keratinocytes appears to be an attractive strategy for improving the therapeutic action of these grafts. The use of epidermal-specific promoters as expression drivers of exogenous genes results in both high expression levels and stratum specificity, as shown in transgenic mice studies. Most current gene transfer protocols to primary keratinocytes involve transduction of epidermal cells with retroviral vectors. However, transfer of gene constructs harboring these long DNA fragment promoters cannot be achieved through viral transduction. In this paper, we describe a protocol consisting of lipid-mediated transfection, G418 selection and an enhanced green fluorescence protein (EGFP)-based enrichment step for obtaining high levels of transgene-expressing primary keratinocytes. Using this protocol, the cDNA for vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocytes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response in the host stroma as determined by fresh tissue examination and CD31 immunostaining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an ‘angiogenic’ composite material may improve wound bed preparation and coverage with cultured keratinocyte grafts.

Introduction

One of the greatest achievements in the treatment of large skin wounds has been the use of graftable autologous cultured epidermal cells.1,2 Grafting of cultured keratinocytes to replace epidermis for skin burns or other cutaneous pathologies often faces the problem of poor take because of infections, hemorrhage and/or impeded production of granulation tissue by the host.3,4,5 The grafted tissue could be viable only if an appropriate source of nutrients and oxygen is provided by a healthy and vascularized stromal bed.6,7 This is evidenced when autologous cultured epidermal sheets are grafted on to a clean dermal bed provided by cadaver skin. This temporary coverage, used for large surface third degree burns, favors graft take as compared with grafting on to an unprepared wound bed. Unfortunately, the demand for allograft skin outstrips supply; in the United States, for instance, by a factor of five to seven.8 To overcome this problem, several artificial skin substitutes are currently being developed as immediate wound coverage.9,10,11,12 In addition to the barrier function of temporarily grafted cadaver skin or composite grafts, allogeneic keratinocytes appear to contribute to the restoration of the host wound bed to which the autologous epidermal graft is to be applied. It is well-established that during wound healing, keratinocytes up-regulate the expression of numerous cytokines that appear to contribute to tissue repair.13,14,15 None the less, seriously damaged dermal tissue may not efficiently sense the stimulus provided by grafted keratinocytes. It is therefore desirable to provide coverage of the wounded area by keratinocytes overexpressing either a particular or various healing-promoting factors, and by a dermal substitute that restores damaged stroma and facilitates neovascularization. In this regard, it has recently been shown that genetically modified human keratinocytes overexpressing platelet-derived growth factor (PDGF) enhance the performance of a composite skin graft.16 The importance of angiogenesis in skin physiology and pathology has recently been highlighted in several reports.17,18,19 The identification and characterization of various cutaneous angiogenic factors now permit the problem of graft neovascularization to be addressed. Keratinocyte-derived vascular endothelial growth factor (VEGF-A), a blood capillary endothelial cell-specific mitogen, is likely to play a major role in the neovascularization associated with the skin wound healing process.15,20 In this regard, the db/db mouse, a model for delayed wound healing, shows reduced VEGF expression levels upon skin wounding.21 To study the role of VEGF overexpression in cutaneous biology, we and others have generated transgenic mice overexpressing a VEGF cDNA driven by keratin gene regulatory elements to target expression of the transgene to the epidermis.22,23 Both studies reported increased skin vascularization, despite the fact that in one case the VEGF cDNA was driven by the basal keratin K14 promoter and in the other, inducible transgenic VEGF expression occurred when the hyperproliferative-responsive keratin K6 promoter was used. These and other transgenic data reinforce the concept that keratinocytes may act as a source of cytokines and/or therapeutic proteins that reach the underlying dermis or the bloodstream to exert biological action.24,25

Ex vivo targeting of keratinocytes for gene therapy has been performed almost exclusively using retroviral vectors.26,27,28,29,30,31 In spite of several advantages of this approach, in vivo maintenance of gene expression driven by the viral long terminal repeat (LTR) promoters has proved difficult for reasons not yet entirely understood.31,32,33 Furthermore, gene constructs harboring long DNA fragments cannot be incorporated within retroviral vectors. On the other hand, nonviral transfer of gene constructs bearing strong, tissue-specific endogenous promoters, such as the 5.2 kb keratin K5 promoter,34 has thus far been hampered by the notorious difficulty for permanent transfection of primary keratinocytes. In this study, we have overcome this limitation using a combination of lipid-mediated transfection, G418 selection and fluorescence-activated cell sorting (FACS) enrichment of primary pig keratinocytes expressing the foreign gene. We have also improved the stromal matrix using a recently developed dermal equivalent based on live fibroblast-containing fibrin gels that allow both efficient keratinocyte growth and graft take.12 This new approach has been successfully applied to achieve a dramatic increase in cutaneous angiogenesis through grafted VEGF- overexpressing primary pig keratinocytes.

Results

Expression vectors

To target VEGF and the enhanced green fluorescent protein (EGFP) cDNA expression to primary keratinocytes ex vivo for subsequent organotypic culture and grafting, we employed gene constructs similar to those previously designed to direct high cell type-and stratum-specific gene expression levels in the skin of transgenic mice.34,35,36 The cDNAs for the mouse heparin-binding isoform VEGF164, the non-heparin binding VEGF120 and EGFP were assembled in an expression vector cassette with the 5.2 kb bovine keratin K5 gene regulatory element, the rabbit β-globin intron 2 and the SV40 polyadenylation signals to aid in the processing of transcripts (Figure 1).

Figure 1
figure1

Scheme of transgene constructs used for keratinocyte transfection. The 5.2 kb fragment of the 5′ regulatory sequences of bovine keratin K5 was used to drive the expression of EGFP, mouse VEGF120 or mouse VEGF164 cDNAs (piled boxes). The cDNA was inserted between the rabbit βglobin intron 2 and the SV40 polyadenylation sequence to enhance gene expression. The arrow indicates initiation of transcription.

Transfection of VEGF and GFP expression vectors into primary pig keratinocytes: selection and enrichment of high level expressing cells

Primary pig keratinocytes were cultured in standard Rheinwald and Green submerged cultures,37 and first passage cells were used for transfection experiments. Cells were contransfected with K5-EGFP and K5-VEGF expression cassettes. All transfections included a neo-resistance expression plasmid. G418 treatment, used as a first selection step, rendered visible resistant colonies after 7–10 days, which were allowed to reach 60–80% confluence in the presence of G418 (7–10 additional days), trypsin detached and pooled for cell sorting. FACS analysis showed that approximately 40–70% of G418-resistant cells were also found to express EGFP (Figure 2a). Transcription of the transfected EGFP and VEGF cDNAs is under the control of the keratin K5 gene regulatory elements. It is therefore, expected that, within the EGFP-positive population, sorting of the cells expressing high EGFP levels (EGFPhigh fraction) will concomitantly enrich for a high VEGF-expressing cell population (VEGFhigh cells). Enzyme-linked immunosorbent assay (ELISA) analysis indeed detected increased VEGF concentrations in the conditioned medium from EGFPhigh cell cultures, as compared with that of unsorted cells (Figure 2b). This strategy enables separation of nontransfected and low-level-expressing keratinocytes from those expressing high VEGF levels.

Figure 2
figure2

FACS analysis and VEGF secretion to conditioned medium of K5-EGFP/K5-VEGF-transfected keratinocytes. (a) EGFP expression in G418-resistant keratinocytes. Resistant colonies were trypsinized, pooled, resuspended and FACS analyzed as described in Materials and methods. Positive and negative populations for EGFP expression were clearly differentiated among the G418-resistant cells. The profile of untransfected keratinocytes is overlaid. (b) Secretion of VEGF by transfected keratinocytes. Conditioned medium of confluent keratinocytes was assayed by ELISA. Right bar, high EGFP-sorted K5-EGFP/VEGF-transfected keratinocytes. Middle bar, unsorted K5-EGFP/VEGF-transfected keratinocytes. Left bar, mock-transfected keratinocytes. The data presented were obtained from three independent experiments and are expressed as pM VEGF per 106 keratinocytes/24 h (mean ± s.d.; n = 3).

After sorting, EGFPhigh/VEGFhigh cells were cultured on a fibroblast-containing fibrin matrix (see Materials and methods) for grafting. To assess whether this matrix influenced transgenic VEGF expression/secretion, sorted keratinocytes were seeded either on 3T3 feeder layers or on fibrin-fibroblast gels. In both cases, the genetically manipulated keratinocytes were successfully expanded to confluence in culture, and conditioned media were assayed by ELISA for the presence of mouse VEGF. The K5-VEGF-transfected keratinocytes produced similar amounts of secreted VEGF when cultured either on feeder layers or on fibrin-fibroblast gels (Figure 3).

Figure 3
figure3

VEGF secretion by transfected keratinocytes growing on different matrices. Sorted EGFPhigh keratinocytes secreted similar VEGF164 levels into the conditioned medium when growing either on 3T3 feeder layer/plastic (closed bars) or on fibrin-fibroblast gels (open bars). The data presented were obtained from three independent experiments and are expressed as pM VEGF per 106 keratinocytes/24 h (mean ± s.d.; n = 3).

Induction of angiogenesis after grafting VEGFhigh keratinocytes

After having achieved efficient and stable mouse VEGF expression in primary keratinocytes in vitro, we examined whether angiogenesis could be induced by these cells after grafting to nude mice. Grafts of keratinocytes transfected with K5-EGFP alone were used as controls. Composite grafts were obtained by allowing keratinocytes to reach confluence on the fibrin-fibroblast gels, followed by subcutaneous transplantation using the flap technique.38 Fifteen to 20 days after grafting, epifluorescent illumination of the dermal side of the flap transplanted area revealed the presence of EGFP-positive tissue. EGFP is a sensitive reporter because it is easily detected, cell-restricted, and stable. To define the localization of transgene expression, frozen skin sections from EGFP-positive areas were evaluated by fluorescence microscopy. Fluorescence was detected almost exclusively in basal cells (Figure 4a), an expression pattern consistent with that previously reported when the same keratin K5 promoter was used in transgenic mice studies.34,35,36 To rule out putative autofluorescence from basal cells we visualized EGFP fluorescence in grafts where EGFP expression was driven by a keratin K6 suprabasal promoter39 (Larcher et al unpublished). No autofluorescent basal cells were detected (Figure 4a, inset). Low power magnification of an area grafted with K5-VEGF-transfected keratinocytes (Figure 4c) showed highly increased vascular density compared with an adjacent untransplanted dermal zone (Figure 4b). Increased magnification revealed the presence of the characteristic VEGF-induced tortuous blood vessels (Figure 4d). These vessels reached an EGFP-positive area, as shown by fluorescence microscopy (Figure 4e) of the same field visualized under transmission microscopy (Figure 4d).

Figure 4
figure4

Transgene expression localization and induction of angiogenesis by VEGF expressing-keratinocyte composite graft. (a) Transversal frozen section of a 15-day K5-EGFP-transfected keratinocyte graft visualized under the green and blue fluorescence channels of the fluorescence microscope (double fluorescence). EGFP (green fluorescence) is expressed in the basal cell compartment of the graft. Nuclei are stained with DAPI (blue fluorescence) and white dashes identify the epidermal–dermal border. Original magnification 200×. The inset (bottom, right) shows the suprabasal expression of EGFP when the EGFP cDNA is under the control of K6 promoter. Note the absence of autofluorescence in basal cells (arrowheads). (b) Stereomicroscope view of a dermal area adjacent to the graft shown in (c). Cutaneous vessels of various diameters cross the field. Original magnification 8×. (c) Stereomicroscope view of the dermal area comprising the K5-EGFP/K5-VEGF keratinocyte graft. Increased vascularization is clearly observed as a dense mesh of blood vessels reaching the graft. The grafted area is indicated by arrowheads. Original magnification 8×. (d) Fresh tissue whole mounts of the grafted area shown in panel c, visualized by white light transillumination microscopy, showing high vascular density. The black dotted lines delimitate the approximate location of the graft. Original magnification 20×. (e) The same field as in panel d under fluorescent epi-illumination shows the EGFP-positive area corresponding to the graft. Note that most blood vessels appear to branch and end at the level of the graft. Original magnification 20×.

As expected, immunoperoxidase staining of CD31/PECAM-1, a specific endothelial cell marker, demonstrated a dramatic increase in the number of blood vessels in the stroma of VEGF-expressing grafts (Figure 5c) as compared to the underlying dermal tissue of normal mouse epidermis (Figure 5a) and control grafts (Figure 5b). Remarkably, and probably as a consequence of the chemoattractant effect reported for VEGF,22,40 capillaries were frequently detected close to the keratinocyte implant, a situation rarely observed in normal mouse skin (compare Figure 5a and c) or in control grafts (K5-EGFP; compare Figure 5a and b).

Figure 5
figure5

Immunoperoxidase staining of blood vessels in grafts of K5-VEGF keratinocytes. (A) CD31 staining of blood vessels in a frozen section of a K5-VEGF graft. Note the high vessel density in the stroma and the proximity of capillaries to the VEGF-overexpressing keratinocytes. (B) CD31 staining of a section of a control (K5-EGFP) graft. Blood vessels, few and scattered, are restricted to a deeper dermal localization as compared with the VEGF-expressing graft. (C) CD31 staining of a section of control mouse skin. Blood vessels of the stroma underlying the normal mouse epidermis present a density and localization similar to the control graft shown in B. Arrowheads indicate capillary vessels. Original magnification ×80.

In spite of the fact that both GFP protein visualization and the strong induced angiogenic response indicated VEGF synthesis, we tested whether transgene transcription driven by a keratin promoter remained active in the graft. Northern blot analysis showed the presence of a 1.0 kb band corresponding to the transgenic VEGF mRNA at 20 days after grafting (45 days after transfection), the longest time of graft analysis, demonstrating continuous expression of the transgene in vivo (Figure 6). The relative contribution of epidermal tissue mRNA to the total RNA sample was determined by keratin K14 mRNA levels. A similar K14/transgenic VEGF mRNA expression ratio was found both in the graft and in the skin of K6sVEGF transgenic mouse, used as positive control, for which a strong angiogenic response has recently been reported.22

Figure 6
figure6

Northern blot analysis of K5-VEGF keratinocyte graft. RNA was extracted from the skin of a K6sVEGF transgenic mouse expressing VEGF (K6s-VEGF mice, see Ref. 19; positive control, lane 1), dermal tissue of a sham-operated mouse (lane 2) and the graft of K5-VEGF keratinocytes (lane 3). Total RNA (15 μg) from each sample were analyzed by Northern blot, using a filter hybridized with a mouse VEGF cDNA probe (upper panel), and subsequently re-hybridized with a human K14 cDNA probe (middle panel) and a 7S cDNA probe to control RNA loading (lower panel).

Discussion

The use of cultured keratinocyte sheets for grafting to severely burned patients was first established in 1981.41 Since then, this technique has been applied to other types of skin lesions.6,9 The epidermal sheets obtained using the standard Rheinwald and Green culture method/dispase detachment must be applied to a healthy dermal bed to achieve appropriate graft take. Indeed, when these grafts are applied on to muscle fascia, they tend to be unstable and blister,7,9 and severely damaged stromal tissue will be unable to nourish such a feeble grafted barrier. To improve the dermal bed, allografted skin is frequently used to temporarily dress the wounded surface. At the time of grafting, the allografted epidermis is removed by shave excision or dermatome and the remaining stroma appears to be suitable for grafting of cultured autologous keratinocytes.6 Cadaver skin allografts have given very good results,42,43 but the availability of such donor skin is limited in comparison with the potential clinical demands. In recent years, considered effort has been invested in developing composite grafts of cultured keratinocytes seeded on to dermal analogues to supply both the epidermal barrier and the stromal bed for temporary wound coverage.6,9 A further improvement envisaged for these bioengineered materials would rely on the use of genetically modified composite grafts able to deliver growth factors or cytokines to improve the dermal bed and enhance wound healing. A recent report describes the augmented performance of a composite graft material in which keratinocytes have been genetically manipulated to overexpress PDGF-A.16

Here, we have explored the possibility of improving the dermal bed by increasing the vascularization through the overexpression of angiogenic factors produced by genetically manipulated keratinocytes. Restoration of vascular tissue should have a major impact on the take and survival of the cultured autologous epidermal graft.

Pig keratinocytes are similar to human epidermal cells in terms of morphology, culture conditions and ability to generate organotypic structures both in vitro and in vivo.28,44 Using these cells, we have been able to introduce stably genetic material by lipid-mediated transfection. A low concentration G418 selection step for 15–20 days gave rise to a G418-resistant population, with 40–70% of these cells expressing high EGFP and VEGF levels, as determined by FACS and ELISA analysis, respectively. This selection/sorting method allowed us to obtain permanently transfected cells with no need for higher G418 concentrations which have been reported to be harmful for keratinocytes in terms of replicating efficiency.45

The unmatched number of neo-resistant and EGFP-positive cells can be explained in several ways. First, the use of independent plasmids in the transfections may result in cell integrating the resistance but not the marker plasmid into their genome. Second, low copy number of co-integrated transgenes may result in G418 resistance, but barely detectable levels of the other transgenes. Third, potential passive transfer of the neomycin resistance gene product could account for the 30–60% G418-resistant cells not expressing EGFP. This last is perhaps a plausible explanation, since G418 selection was carried out with low drug concentrations on transfected cells without splitting.

We recently demonstrated that constitutive overexpression of VEGF120 driven by a suprabasal K6 keratin promoter in transgenic mouse epidermis has a dramatic effect on cutaneous vascularity. A similar transgenic mouse study using VEGF165 and the keratin K14 regulatory elements also showed increased vascularization, although to a lesser extent.23 Using similar gene constructs to those employed in the above-mentioned transgenic studies, we manipulated primary keratinocytes ex vivo. After grafting, these cells elicited an angiogenic response that closely resembled that observed in vivo. A remarkable feature of both the K6-VEGF transgenic and the ex vivo modified keratinocytes is the chemoattractive capacity for blood vessels of the VEGF overexpressing epidermal tissue. From the point of view of the temporary improvement of vascularization of damaged stroma, the rapid recruitment of capillaries to the vicinity of the permanent keratinocyte autograft may have beneficial properties.

Studies using transgenic mice in which factor IX and growth hormone genes were driven by keratin K10 and K14 promoters, respectively, have indicated the potential utility of epidermal cell endogenous promoters for gene therapy.24,25 Our study is the first to demonstrate that targeting to keratinocytes, using lipid-mediated transfection of plasmid constructs containing endogenous promoters as well as the expansion and subsequent grafting of these cells for gene therapy, is a feasible approach. Expression driven by regulatory sequences of genes coding for structural proteins such as keratins, involucrin or fillagrin ensures precise stratum localization and abundant production of the therapeutic protein. Previous studies by our group also indicated that transgene expression can be modulated using the inducible keratin K6 promoter.22,39 Proper function of these regulatory sequences in transgenic mice requires several kb long DNA fragments, precluding their use in the construction of retroviral vectors.

Additional studies using animal models (ie full-thickness skin wounds in pigs) are required to assess the actual advantages that an ‘angiogenic’ composite graft might provide for the permanent closure of large wounds. Given the similarities between pig and human keratinocytes, it is conceivable that the conditions can be established to achieve permanent expression of transgenes, through tranfection, in human epidermal cells. Although experiments are under way, we have not yet succeeded in duplicating this approach in primary human keratinocytes. Whether human keratinocytes are more susceptible than pig keratinocytes to the cytotoxic effects of G418, or present a lower gene integration efficiency are phenomena currently under investigation.

Our approach offers the possibility of giving patients suffering from large skin trauma/defects, an optimized composite graft able to act not only as an elaborate dressing but also as a strong stimulator of vascularization. Although the modified keratinocytes would gradually be replaced by those of the host or by subsequent cultured autologous grafts, these would face a ‘ready-to-go dermis’. Potential banking of angiogenic composite grafts may offer a prospect for immediate wound coverage.

Materials and methods

Plasmids

The VEGF cDNAs were excised as EcoRI fragments from plasmids pVEGF1 and pVEGF2.46 The EGFP cDNA was excised as an EcoRI–NotI fragment from plasmid pEGFP–N1 (Clontech, Palo Alto, CA, USA). Each cDNA was introduced in the polylinker of vector p163/7,47 as previously described.22 The SalI–KpnI fragment containing the 5′β-globin intron 2, the cDNAs and the 3′ polyadenylation sequences were inserted 3′ downstream of the 5.2 kb fragment of the bovine K5 regulatory sequences34 cloned in pBluescript.

Cell culture and fibrin-fibroblast gel preparation

Keratinocytes derived from Yucatan minipig skin (age 4–8 weeks) were isolated following previously described methods.48 Cells (2 × 106) were cultured with lethally irradiated 3T3-J2 fibroblasts (2 × 106) on 75 cm2 culture flasks. The culture medium was a 3:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL, Life Technologies, Barcelona, Spain) and Ham-F12 (GibcoBRL) supplemented with 10% fetal calf serum (FCS) (BioWhittaker, Wallkersville, MD, USA), insulin (5 mg/ml; Sigma, St Louis, MO, USA), hydrocortisone (0.4 mg/ml; Sigma), cholera toxin (8 ng/ml; Sigma), EGF (10 ng/ml; Sigma) and adenine (24 mg/ml; Sigma). The medium was changed every 2 days, and the cultures were incubated in a humidified 5% CO2 atmosphere at 37°C. When keratinocyte primary cultures were 70% confluent, they were treated with trypsin/EDTA to obtain individual cells. Cells were then subcultured on 3T3-J2 fibroblasts or frozen for later use.

Fibrin-fibroblast gels were prepared as previously described12 for the culture of human keratinocytes, with modifications. Pig fibroblasts (2 × 104/cm2), used instead of human fibroblasts, were isolated from pig skin biopsies by enzymatic digestion and cultured in DMEM containing 10% FCS. They were used in the fibrin-containing gels between passages four and 12. Fibrinogen from pig blood plasma cryoprecipitates was used as the fibrin source. Cryoprecipitates were obtained according to the standards of the American Association of Blood Banks.49 Each unit of cryoprecipitate was heated at 37°C for 45–60 s, and before complete thawing, it was centrifuged (2800 g, for 15 min, 4°C). The supernatant was discarded, the pellet dissolved in 10 ml of saline, then, heated to 37°C to complete fibrinogen dissolution. Approximately 150–300 mg of fibrinogen were obtained from each unit of cryoprecipitate. To produce a fibrin gel, 3 ml of the fibrinogen (cryoprecipitate) solution were added to 12 ml of DMEM/10% FCS containing 5 × 105 pig fibroblasts and 5000 IU of bovine aprotinin (Trasylol, Bayer, Barcelona, Spain). Immediately afterwards, 1 ml Cl2Ca (0.025 mM; Sigma) with 11 IU of bovine thrombin (Sigma) was added. Finally, the mixture was seeded in a 75 cm2 culture flask and allowed to solidify at 37°C. The gel was covered with culture medium and used 24 h later.

Transfected or transfected-sorted keratinocytes were seeded on irradiated 3T3-J2 cells (a gift of Dr M Simon, Living Skin Bank, SUNY, Stony Brook, NY, USA) or on fibrin-live fibroblast gels at a density of 2–3.5 × 105 cells/25 cm2.

Transfection experiments and FACS analysis of EGFP-expressing cells

For transfection experiments, 5 × 105 keratinocytes were plated per 100 mm dish on irradiated neo-resistant 3T3-J2 fibroblasts (a gift of Dr J Garlick, SUNY, Stony Brook, NY, USA). After 4–7 days in culture, the cells were cotransfected using lipofectamine (GibcoBRL Life Technologies), with the plasmids K5-EGFP, K5-VEGF (the latter plasmid was not included in the transfection of keratinocytes used for control grafts), and the neo-resistance containing plasmid 9004,50 in which the neo sequence is flanked by the adeno-associated virus inverted terminal repeats (ITRs). The presence of ITRs was shown to increase the integration frequency of flanked reporter sequences, both in 293 cells50 and in the human epidermal cell line HaCaT, as compared with the neo resistance plasmid pcDNA3 (Del Rio et al, unpublished observation). Keratinocytes were transfected with 30 μg of plasmid DNA (20 μg K5-VEGF, 5 μg K5-EGFP and 5 μg 9004) complex in serum-free medium (OptiMem; Gibco) with 30 μl of lipofectamine. At 48 h after transfection, G418 was added at a final concentration of 0.3 mg/ml. The medium was changed every 4 days for a 14–20 day selection period. Resistant cells at 60–80% confluence were trypsinized, resuspended in PBS/2% FCS, analyzed for EGFP expression and sorted by fluorescence-activated cell sorting (FACS) on a FACStar PLUS (Becton Dickinson, San Jose, CA, USA) flow cytometer.

Detection of mouse VEGF in transfected keratinocyte-conditioned medium

Mouse VEGF protein concentration in keratinocyte-conditioned media was quantified by ELISA using a commercial kit and following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Measurements were performed for keratinocytes transfected with the cDNA for mouse VEGF164, which is the isoform recognized by the kit.

Grafting to nude mice

At keratinocyte confluence, cultures on fibrin-fibroblast gels were manually detached, divided into 4 cm2 squares, and grafted on to the dorsal region of athymic mice (one square per mouse) following the flap method previously described by Barrandon et al.38 VEGF/EGFP transfected-sorted keratinocytes or EGFP transfected-sorted control keratinocytes were grafted on to five to six mice. Mice were housed in pathogen-free conditions for the duration of the experiment. Most grafts were harvested within 15–20 days after grafting, and tissue specimens were frozen to perform immunostaining and Northern blot, or maintained on ice until direct visualization of EGFP fluorescence.

Visualization of EGFP in fresh tissue and tissue sections

To determine the precise localization of the graft, a piece of back skin comprising the graft was excised from transplanted mice after they were killed, whole-mounted on a glass slide with the dermal side up and observed at low power magnification (×10) using the fluorescein isothiocyanate (FITC) channel of a Zeiss (Oberkochen, Germany) fluorescence microscope. Green fluorescence was indicative of EGFP-positive tissue. EGFP was also visualized in 1% paraformaldehyde-fixed (7 min, 4°C) transversal frozen sections of the EGFP-positive areas identified in the fresh tissue.

Detection of blood vessels

Blood vessels were visualized in fresh graft-containing skin samples by both epi-illuminated stereomicroscopy and transilluminated, low power magnification microscopy using white light. Vascularization was also studied by immunostaining of frozen sections using the CD31/PECAM-1 antibody (PharMingen, San Diego, CA, USA) as previously described.22

Northern blot analysis

Total RNA was extracted from GFP-positive grafted areas using Trizol reagent (GibcoBRL). Only the graft and the underlying stromal tissue were excised, carefully excluding mouse epidermal tissue. Whole skin from K6sVEGF120 transgenic mouse22 was used as a positive control for transgenic VEGF expression. RNA (15 μg per lane) was analyzed by Northern blot hybridization as described,22 using 32P random-primer labeled probes specific for mouse VEGF,40 human keratin K14 or 7S ribosomal RNA.

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Acknowledgements

This work was supported by a grant from the Comunidad Autonoma de Madrid (CAM). We thank I de los Santos, S Moreno and I Orman for expert technical assistant and Cathy Mark for editorial revision of the manuscript. MDR is a recipient of a postdoctoral fellowship from CAM 02/0078.

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Correspondence to J L Jorcano.

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Del Rio, M., Larcher, F., Meana, A. et al. Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter. Gene Ther 6, 1734–1741 (1999) doi:10.1038/sj.gt.3300986

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Keywords

  • gene targeting
  • lipofection
  • keratin promoter
  • keratinocytes
  • angiogenesis
  • VEGF

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