Inducible expression of green fluorescent protein in porcine tracheal epithelial cells by the bovine tracheal antimicrobial peptide promoter

Inducible Expression of Green Fluorescent Protein in Porcine Tracheal Epithelial Cells by the Bovine Tracheal Antimicrobial Peptide Promoter Paul W. Dyce, Robert J. DeVries, John Walton, Roger R. Hacker, Julang Li Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1; telephone: 519-824-4120, ×52713; fax: 519-836-9873; e-mail: [email protected] Received 17 March 2003; accepted 4 June 2003 Published online 18 August 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10779

Abstract: Tracheal antimicrobial peptides (TAP) are expressed primarily in respiratory epithelial cells of cattle. The TAP expression is inducible upon challenge with bacteria and bacterial lipopolysaccharide (LPS). In pigs, a promoter that can be activated by bacterial infection has yet to be identified. The objective of this study was to use green fluorescent protein (GFP) as a reporter gene to determine the function and inducibility of the bovine TAP promoter in porcine primary tracheal epithelial cells. Thus, evaluating the feasibility of using this promoter to direct transgene expression in porcine cells. The percentage of GFP expressing cells increased in response to LPS challenge in both a dose-dependent and time-dependent manner (p < 0.05). Moreover, when the intensity of the GFP fluorescence was measured, it was observed that the percentage of cells that have a high intensity of GFP fluorescence, also increased gradually as LPS dose increased, the difference between the unchallenged (control) and challenged group become statistically significant at the concentration of 100 ng/mL after 36 h LPS challenge (p < 0.05). The level of inducedexpression driven by the TAP promoter was 67.8 ±12.2% that of the cytomegalovirus (CMV) promoter. The intensity of GFP fluorescence by the TAP promoter was 39.8 ± 7.6% when compared to the expression driven by the CMV promoter. These data suggest the TAP promoter functions at a lower, but comparable, level to the strong CMV promoter. Our data demonstrated that the bovine TAP promoter was functional in porcine primary tracheal epithelial cells. The ability of the TAP promoter to control gene expression in an inducible manner in the porcine respiratory tract presents an important application potential in transgenic animal studies. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 374–381, 2003.

Keywords: tracheal antimicrobial peptide promoter; inducible expression; GFP; porcine; tracheal epithelial cells

Correspondence to: Julang Li Contract grant sponsors: Natural Sciences and Engineering Research Council (NSERC); Agriculture and Agri-Food Canada; Ontario Ministry of Agriculture and Food; Ontario Pork; The Food System Biotechnology Centre at the University of Guelph

© 2003 Wiley Periodicals, Inc.

INTRODUCTION Controlled expression of a transgene is critical for the production of transgenic animals with either enhanced growth performance or improved disease-resistance. Constitutive promoters such as that of cytomegalovirus (CMV) have been used successfully to achieve a high level expression of transgenes (Hoeflich et al., 1999; Hoeflich et al., 2001; Ray et al., 2001). However, inappropriate constitutive expression of a transgene can lead to deleterious or lethal effects (Li and Hoyle, 2001). The ability to control transgene expression in a temporal and spatial manner is important if the goal is to target specific physiological events without deleterious side-effects. By controling the time and tissue in which transgenes are expressed, an inducible expression system can abate deleterious and abnormal effects that can result from untimely or nonlocalized gene expression. Also, certain genes must be expressed at specific stages of development or they can lead to a halting of embryo development (Li and Hoyle, 2001). Ideally, the inducible transgene would react to induction in a dose-dependent manner with the potential for high maximal expression and a low basal expression. Several inducible promoter systems have been described in a variety of species. For example, cecropin is one of four major families of antimicrobial peptides found in insects. The cecropin promoter has been shown to be inducible upon lipopolysaccharide (LPS) challenge in channel catfish (Zhang et al., 1998). The GAL1 promoter in Saccharomyces cerevisiae is repressed by the presence of glucose and induced by galactose in the absence of glucose (Li et al., 2000). The rat heat shock protein (HSP) 70 promoter has been shown, in mice, to induce expression of its gene product upon heat shock (Wysocka and Krawczyk, 2000). The ethanol inducible promoter from the fungus Aspergillus nidulans, alcA, is functional in Arabidopsis thaliana (Roslan et al., 2001). The tracheal antimicrobial peptide (TAP) gene is expressed primarily in respiratory epithelial cells of cattle

(Diamond et al., 1993). Expression of TAP is inducible upon challenge with bacteria and bacterial LPS (Diamond et al., 1996). Further study has demonstrated that expression of the TAP gene is controlled at the transcriptional level by the TAP promoter (Diamond et al., 2000). The TAP promoter was first isolated from bovine tracheal mucosa (Diamond et al., 1991), and the DNA sequence of the TAP gene including the promoter region has also been determined (Diamond et al., 1993). Previous study on the characterization of the TAP promoter in bovine tracheal epithelial cells indicated that sequences within 324 nucleotides of the transcription start site are responsible for driving induced TAP expression. This region consists of consensus binding sites for NF-␬B and nuclear factor interleukin-6 transcription factors (Diamond et al., 2000). The TAP promoter is a tissue-specific promoter that drives expression primarily in the epithelial cells lining the bovine respiratory tract. The tissue-specificity and the inducible features of the TAP promoter, in particular, its nature for responding to bacterial products, represents an important biotechnological application potential. Green fluorescent protein (GFP) has been widely used as a reporter gene since being cloned in 1992 (Prasher et al., 1992). Green fluorescent protein is a reporter protein of 27 kDa, fluorophore spontaneously forms intracellularly without the need for sacrificing or fixing the host cells or the addition of chemical substrates (Chalfie et al., 1994). It is highly fluorescent with a quantum efficiency of 0.85 that absorbs light at 488 nm and emits green fluorescence at 509 nm (Chalfie et al., 1994). Green fluorescent protein fluorescence can be detected at the level of the single cell, without the addition of exogenous substrates. Cells expressing GFP remain viable and positive cells can be sorted using flow cytometry. The GFP is also relatively stable, providing an accurate measure of total gene expression (Ehrmann et al., 2001). Green fluorescent protein has been successfully used for monitoring gene expression in vivo, in vitro, and in real-time assays (Chalfie et al., 1994; Gagneten et al., 1997; Wysocka and Krawczyk, 2000). One important and stimulating aspect when considering transgenic farm animals is the possibility for improving their resistance to disease. An inducible and tissue-specific promoter is desirable for appropriate synthesis of gene products that would help to fight bacterial infection. In pigs, a promoter that can be activated by bacterial infection of the trachea and lungs has yet to be identified. The objective of this study was to use GFP as a reporter gene and determine the inducibility of the bovine TAP promoter in porcine primary tracheal epithelial cells, and to evaluate the feasibility of using this promoter to direct transgene expression in porcine cells. MATERIALS AND METHODS Vector Construction An expression vector was constructed and designated pTAP-EGFP for use in this study (Fig. 1). The GFP se-

Figure 1. Schematic depiction of the pTAP-GFP construct used in the promoter analysis study.

quence and plasmid backbone originated from pEGFP-N1 (Clontech Laboratories, Palo Alto, CA). The TAP promoter sequence (from −380 to 30; according to sequence in GenBank #L13373) was amplified from bovine DNA by PCR using forward primer 5⬘-GCATTAATACT-AGTGGGCACTTTCAAAGCC-3⬘ and reverse primer 5⬘-ATGGATCC-TGCTGGCGTCCCGAGCTCTG-3⬘ (Diamond et al., 2000). Restriction enzyme sites for AseI and SpeI were added in tandem to the 5⬘ end of the forward primer. A BamHI restriction enzyme site was added to the 3⬘ end of the reverse primer to facilitate cloning in the correct orientation. The CMV promoter of pEGFP-N1 was removed by AseI and BamHI digestion and the TAP sequence from PCR was ligated in its place using the same restriction enzyme sites. The resulting plasmid was electroporated into DH5␣ using a BTX ECM399 electroporator (voltage 2.0 kV). The correct insertion of the TAP promoter into the construct was confirmed by restriction enzyme digest and sequencing. Isolation and Culture of Porcine Primary Tracheal Epithelial Cells Tracheas were obtained through local Ontario meat processing plants. The tracheal isolation procedure was adapted from previous reports (Tesfaigzi et al., 1990; Wu and Smith, 1982; Yohannan et al., 1999) with modifications. The tracheas were excised from the respiratory tract immediately following their removal from the pigs. The intact trachea from the larynx to the first bifurcation was obtained and placed into phosphate buffered saline (PBS) solution, containing antibiotic–antimycotic (penicillin 100 IU/mL, streptomycin 100 ug/mL, and amphotericin 250 ng/mL, Invitrogen). The tracheas were then transported to the laboratory, within one hour following their removal, where they were washed three times with PBS, containing antibiotic– antimycotic. The tracheas were then filled with proteinase solution (0.2% dispase (Sigma) in Ham’s F12 (Invitrogen), both ends clamped, and incubated at 37°C for 1.5 h. Following this incubation, the proteinase solution containing mucous was released and discarded, and replaced with fresh proteinase solution for incubation at 4°C overnight. Incubated proteinase solution was then collected from the tracheas and cells pelleted at 500g and washed. The cells were then resuspended in culture medium [1:1 ratio of Ham’s F12

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and DMEM H16 (Invitrogen), 5% fetal bovine serum, 1× insulin-transferin-selenium (Invitrogen)] and plated, at an initial density of ≈ 5 million cells per 10-cm dish, on collagen (Type I, 6 ug/cm2; Sigma, St. Louis, MO) coated plates. The plated cells were incubated at 37°C with 5% CO2 and 95% air overnight, and then the medium was changed. When the cells reached 70–80% confluence, 2–4 d, they were trypsinized and subcultured. Primary Fibroblast Isolation and Culture Primary fibroblasts were isolated according to a previous published protocol (Boquest et al., 1999) with modifications. Briefly, pig fetuses 25 days postcoitum were washed with PBS, the heads and inner organs were excised. The remains were pooled, minced into small pieces (about 1 mm2). The tissue pieces (100 pieces) were then resuspended in 5 mL of Dulbecco’s Minimun Eagle’s medium (Invitrogen) with 5%FBS were used to seed on a 10-cm dish. The explants were removed at day 3 and the remaining monolayer of fibroblasts was harvested by trypsinization. Cells were subsequently subcultured and passaged a few times before transfection. Granulosa Cell Isolation and Culture Gilt ovaries were collected at a local slaughterhouse, and granulosa cells were isolated using techniques described previously (Manikkam et al., 2002) . Briefly, follicles were aspirated by hand, using a 19-gauge needle attached to a 10-cc syringe. After removal of oocytes, granulosa cells were separated from follicular fluid by centrifugation at 500g for 5 min. Cells were washed five times with PBS containing penicillin (50 U/mL), and streptomycin (50 g/mL). Granulosa cells were then cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 g/mL). PK-15 Cell Culture PK-15 cell is a porcine kidney epithelial cell line from American Type Culture Collection. PK-15 cells were cultured in DMEM containing 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 g/mL). The plated cells were incubated at 37°C with 5% CO2 and 95% air. When the cells reached 80% confluence, they were trypsinized and subcultured. Immunocytochemistry The immunocytochemistry was performed according to a previously described protocol (Sweat et al., 2001) with modifications. Briefly, sterilized glass coverslips were placed in culture plates prior to cell plating. Cells were grown on the coverslips to subconfluency. After removing

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the culture medium, the monolayer was rinsed two times in PBS and fixed in 100% methanol for 10 min at room temperature. After rinsing with PBS three times, the fixed cells were permeabilized with 0.1%Triton X-100 in PBS for 2 min. The cells were washed three times in PBS (10 min/ each), blocked in 10% bovine serum albumin (BSA fraction V; Sigma) for 10 min and then incubated with monoclonal anti-cytokeratin antibody (Sigma; 1:200 dilution in PBS with 1% BSA). After an 18 h incubation period at 37°C, the plates were incubated at 4°C for 30 min to stabilize the antibody–antigen binding. The cells were washed and blocked again as previously described. They were then incubated in goat anti-mouse IgG FITC conjugate (1:200 dilution in PBS) for 1 h at room temperature in the dark. After washing, the coverslips were placed on microscope slides for observation under a UV microscope. Gene Delivery 5 × 106 cells were resuspended in 360 uL of electroporation buffer (75% cytosalts (120 mM KCl, 0.15 CaCl2, 10 mM K2HPO4, 5 mM MgCl2, pH 7.6, and 25% Opti-MEM (Invitrogen) in a 4-mm cuvette. The samples were then mixed with 10 ug of pTAP-EGFP, and electroporated using the Bio-Rad gene pulser II electroporator. The electroporation settings were 480 volts, 25 uF capacitance, four bursts, with a duration of 1 ms. The negative control was electroporated without the presence of a vector. The positive control consisted of 10 ug of pCMV-EGFP. Immediately following electroporation the samples were placed on ice for 5 min. One milliliter of cell culture medium was then added to the electroporated samples and they were incubated at 37°C for 10 min. The pTAP-EGFP vector electroporation was plated on collagen-coated plates (1 × 106/plate; 6 cm in diameter). The negative and positive controls were plated at the same density on single plates and incubated at 37°C and 5% CO2. Posttransfection Challenge The electroporated cell cultures were incubated for 10–12 h allowing for the cells to attach to the plates, and the medium was changed to remove suspended (dead) cells. LPS was then added to the five plates in the following concentrations: 0, 10, 50, 100, and 150 ng/mL and incubated for 20 or 36 h prior to being analyzed by flow Cytometry. Quantification of Expression Level by Flow Cytometry Following the LPS challenge the cells were detached from the dishes using trypsin-EDTA. The cells were washed and then resuspended in PBS at a concentration of 0.5 million cells/mL. The cells were analyzed on a FACScalibre (Becton Dickenson, San Jose, CA) flow cytometer. Analysis was performed using CELLQuest Version 3.1f software (Becton Dickenson Immunocytometry systems). GFP expression

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was detected using an argon laser that produces at a wavelength of 488 nm. The FL1 photomultiplier tube (PMT) detected the green fluorescence emitted from the GFP. Gates were set to exclude necrotic cells and cellular debris and the fluorescence of events in the selected region was quantified. Data were collected from 10,000–20,000 events for each sample. The negative control sample was used to set a gate such that no more than 0.07% of these cells (auto fluorescing cells) fell into the positive range. Statistics Experiments were replicated three to five times. Statistical analysis of the data was performed using Prism Software (Prism software version 3.0; GraphPad, San Diego, CA). The dose response and time course data were analyzed using a one-way ANOVA followed by a Tukey test. Statistical significance was inferred at p < 0.05. RESULTS Establishment of and Characterization of Primary Porcine Tracheal Epithelial Cells In Vitro One of the challenges of working with a livestock cell model is the lack of an appropriate cell line. Although the isolation method of porcine tracheal epithelial cells was described previously (Tesfaigzi et al., 1990; Yohannan et al., 1999), the purity of the cells and the culture potential were not determined in those studies. The protocol from this and other species (Tesfaigzi et al., 1990; Wu and Smith, 1982; Yohannan et al., 1999) was modified to isolate porcine tracheal epithelial cells. With our optimized culture system, porcine tracheal epithelial cells were cultured for up to four passages, after which significant morphological changes were observed, including increased cell diameter and a reduction in growth rate. Cytokeratins are well-established characteristics of epithelial cells (Adams and Watt, 1988; Lane and Alexander,

1990; Moll et al., 1982). To determine the purity of the cells in our isolation and culture system, immunocytochemistry with monoclonal anti-pan cytokeratin antibody was performed. As shown in Figure 2, greater than 90% of the cultured cells expressed cytokeratins, suggesting a majority of the cells were of epithelial origin. Increase of TAP Promoter Activity in Response to LPS in a Dose-Dependent Manner LPS is a component of the outer membrane in gramnegative bacteria. LPS has been successfully used in previous studies, to simulate bacteria infection, without the risk of using viable pathogens (Diamond et al., 1996; Zhang et al., 1998). As shown in Figure 3 (upper panel), the percentage of GFP expressing cells increased in response to LPS challenge in a dose-dependent manner (coefficient of linearity ⳱ 0.0245, p < 0.0001). Moreover, when the intensity of the GFP fluorescence was measured, it was observed that the percentage of cells that have a high intensity of GFP fluorescence, also increased gradually as the LPS dose increased. The difference between the unchallenged (control) and challenged group became statistically significant at the concentration of 100 ng/mL LPS (Fig. 3 lower panel). This data suggests that the TAP promoter could not only be functional, but also its activity be further up-regulated by LPS in porcine tracheal epithelial cells. Time-Course Study and the Level of Induction Since 150 ng/mL of LPS induced TAP promoter activity in the dose-response study, this dosage was chosen to study the time required to induce TAP- driven GFP expression. Twelve hours posttransfection, the transfected cells were further cultured for 20, 36, and 48 h in the absence and presence of LPS (150 ng/mL), respectively. Flow cytometry analysis indicated that the percentage of both the total GFP expressing cells and cells which express GFP at a high level appeared to increase following 20 h of exposure to LPS, but the difference between the control and the challenged group

Figure 2. Expression of cytokeratins in porcine tracheal epithelial cells. Tracheal epithelial cells were grown to sub-confluence on coverslips; immunocytochemstry was performed using anti-pan cytokeratin antibody and a secondary antibody with an FITC conjugate. (A) Cultured porcine tracheal epithelial cells under normal light on an inverted microscope (200× magnification). (B) The same tracheal epithelial cells under UV light (200× magnification).

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Figure 3. Concentration-dependent increase of TAP-driven GFP expression induced by LPS in vitro. Twelve hours after transfection with the pTAP-GFP construct, the tracheal epithelial cells were challenged with various concentrations of LPS (0–150 ng/mL) for 36 h. Cells were then trypsinized and analyzed for GFP expression by flow cytometry. Upper panel, total GFP expressing cells; Lower panel, GFP highly expressing cell. Data are expressed as the percentage of control (100%) and represent mean ± SEM of three experiments. *, p < 0.05; **, p < 0.01, compared to control.

was not statistically significant (Fig. 4). Following a 36-h challenge, the number of both total GFP expressing cells and GFP highly expressing cells increased significantly (p < 0.01), with the increase of the later being more dramatic (p < 0.01; 350% compared to control; Fig. 4A, 4B). A representative example of the flow cytometry data from the 36 h challenge study is shown in Figure 5. At 48 h posttransfection, no further increase in GFP expression was observed (data not shown). The level of induction of the TAP promoter in tracheal epithelial cells was also studied. Since the efficiency of transfection in primary cells is extremely low and variable a parallel system was used to provide a reference for determination of the promoter activity. In each of the experiments, a control transfection was done with the same amount of the pCMV-GFP constructs, which was exactly the same as pTAP-GFP except the TAP promoter was replaced with the CMV promoter to monitor the transfection efficiency and as a control of the expression level. The comparison of CMV- and LPS induced TAP driven GFP expression is shown in Table I.

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Figure 4. Time-course studies on the induction of TAP-driven GFP expression in tracheal epithelial cells by LPS. Tracheal epithelial cells were transfected with pTAP-GFP. After 12 h of pre-plating time, the transfected cells were further cultured in the absence and presence of LPS (150 ng/mL) for up to 36 h. Cells were then trypsinized and analyzed for GFP expression by flow cytometry. Upper panel, total GFP expressing cells; Lower panel, GFP highly expressing cell. Data are expressed as the percentage of control (100%) and represent mean ± SEM of five experiments. **, p < 0.01, compared to control.

Specificity of the TAP Promoter in Porcine Cells In cattle, the TAP promoter exhibits activity mainly in tracheal epithelial cells (Diamond et al., 1993). In our study, porcine primary fibroblast cells were used in the place of the tracheal cells to test whether the TAP promoter retains its tissue specificity in the porcine species. Irrespective of the presence and absence of the LPS, the expression of GFP in these transfected cells was not observed (data not shown). In a parallel experiment using pCMV-GFP as a positive control of the transfection, ≈ 15% of the transfected fibroblasts expressed GFP. Similar results were also observed in a tranfection study with porcine granulose cells (data not shown). To study whether TAP promoter is functional and inducible in other porcine epithelial cells, a transfection was also performed with kidney epithelial cell line (PK-15). Approximately 1.5% of the transfected cells expressed GFP at low intensity, and the expression did not change in response to LPS challenge.

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Figure 5. Detection of GFP expression in porcine tracheal epithelial cells by flow cytometry. Tracheal epithelial cells were transfected with pTAPGFP. After 12 h of pre-plating, the transfected cells were further cultured in the absence and presence of LPS (150 ng/mL) for up to 36 h. Cells were then trypsinized and analyzed for GFP expression by flow cytometry. (A) Transfected cells without LPS challenge; (B) Transfected cells challenged with LPS (150 ng/mL) for 36 h.

DISCUSSION To our knowledge, this is the first report on the functionality of an inducible promoter in porcine primary cells. In this investigation, we demonstrated that the TAP promoter is functional when transfected into porcine tracheal epithelial cells. The TAP promoter activity, as indicated by GFP as a reporter gene product, increased in response to the LPS challenge in a dose- and time-dependent manner. The TAP promoter is believed to be a component of the bovine host immune system (Diamond et al., 2000). It has been shown to be inducible by pathogens as well as bacteria products, such as LPS (Diamond et al., 2000). CD14 is a major mammalian receptor for LPS. It is suggested that LPS interacts with CD14 leading to the activation of several transcription factors including NF-␬B. NF-␬B binding activity has been demonstrated in the TAP promoter and the

activity increased upon LPS challenge . In the same study, serum has also been shown to augment TAP induction by LPS (Diamond et al., 2000). The soluble form of CD14 and LPS binding protein (LBP) are present in serum. LPS can complex with LBP and then interact with soluble CD14 to form LPS-sCD14, which possibly interacts with toll-like receptors (TLR), present on epithelial cells (Cario et al., 2000). This then proceeds, as with the membrane bound CD14, to activate the transcription factor NF-␬B. CD14 has been located on the surface of porcine epithelial cells (Liu et al., 2001). Also porcine epithelial cells have been shown to respond to LPS by up-regulating various IL cytokines expression (Liu et al., 2000; Muneta et al., 2002), suggesting that the signaling pathway(s) for LPS is present in the porcine epithelial cells. It is possible that the response of TAP promoter activity to LPS in our study also employed this pathway, since cells were cultured in the presence of serum. The .5-fold (total GFP expressing cells) and 2.5-fold (GFP highly expressing cells) increase in the reporter gene expression in response to the LPS challenge in pig tracheal epithelial cells is comparable to the level of induction in the bovine tracheal epithelial cells where the relative luminescence was increased by onefold in response to the LPS challenge, using luciferase as a reporter gene (Diamond et al., 2000). In addition, the level of GFP expression upon LPS induction was comparable, although lower, to that of GFP expression driven by a strong mammalian cytomegalovirus promoter. In native bovine tracheal epithelial cells, the tracheal antimicrobial peptide mRNA level was increased by as much as 13-fold upon challenge with LPS (Diamond et al., 1996). The fact that the transfection efficiency was 5–20%, together with the fact that transfected cells were not enriched via antibiotic selection may explain the less dramatic increase in induced-expression compared to the native system in bovine tissue. Very few studies on the inducible promoter activity in vertebrate animal cells using the GFP reporter system have been reported. An investigation on channel catfish cells using the insect cecropin promoter demonstrated ≈ 1% increase in GFP-expressing cells and ≈ 50% increase in green fluorescence intensity in response to LPS challenge (Zhang et al., 1998). Thus, the inducibility of the TAP promoter appears to be stronger than the cecropin promoter when functioning in heterogeneity species. The level of unchallenged, basal expression is also important to the utility of an inducible expression system. In the unchallenged group in our experiments 4.27 ± 0.75% of cells expressed GFP (most at a low level of expression). Due to the nature of induction, part of the basal level of expression may be accounted for by the presence of bacterial contaminants in the culture. Although our culture medium contained antibiotics, it is known that bacteria products (part of them is LPS), as well as dead bacteria could by themselves activate the TAP promoter (Diamond et al., 2000). The variability of the basal expression suggests that a low basal level is, however, attainable. In the current study with porcine tracheal epithelial cells,

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Table I. Comparison of CMV- and LPS induced TAP-driven GFP expression in porcine tracheal epithelial cells.

Experiment

Promoter

Total GFP expressing cellsa (%)

1

CMV

8.48

TAP/CMV (%)

GFP highly expressing cellsa (%) 1.36

91.86 2

TAPb CMV

7.79 8.48

3

TAPb CMV

7.64 2.70

4

TAPb CMV

2.17 21.62

5

TAPb CMV

8.19 21.62

TAPb

8.33

38.97 0.53 1.36

90.09

45.59 0.62 1.77

80.37

65.54 1.16 6.34

37.88

23.98 1.52 6.34

38.53 Mean ± SE

TAP/CMV (%)

25.08 1.59

67.75 ± 12.22

39.83 ± 7.63

a The expression of GFP was analyzed with flow cytometry 36 h after transfection with the respective promoter driven expression constructs. b Cells transfected with the TAP-driven construct were challenged with LPS (150 ng/mL).

the response of the TAP promoter to LPS challenge was not as rapid as that in the bovine tracheal epithelial cells (36 h vs. 18 h; Diamond et al., 2000). This difference may have partially resulted from different reporters being used in the promoter activity assay (luciferase vs. the GFP in the current investigation). It was shown in a study using E. coli that there was a time lag between the expression of the GFP determined by Western blot and the emission of its fluorescence (Albano et al., 1996). The fluorescence of GFP is due to a chromophore formed via a post-translational cyclization reaction and subsequent oxidation of three amino acids (Cody et al., 1993; Prasher et al., 1992). The completion of this multiple step process results in a time lag between the protein translation and visible fluorescence (Heim et al., 1994). The other possibility for the difference in lag time may be attributed to species difference. One of the critical tools in the field of transgenesis is the development of an effective gene expression system. A regulated-expression system would allow the time and location of gene expression to be precisely controlled. Geneswitches are especially valuable when working with genes where inappropriate expression is undesirable. These results demonstrated that the TAP promoter is functionally active, and responsive to LPS in porcine tracheal epithelial cells. The level of expression detected appears to be proportional to the LPS challenge, potentially providing a means of endogenously controling the level of gene expression in response to LPS in an in vivo situation. The level of TAP promoter driven induced-expression was 67.8 ± 12.2% that of the CMV promoter. The intensity of GFP fluorescence by the TAP promoter was 39.8 ± 7.6% when compared to the expression driven by the CMV promoter. This data suggests the TAP promoter functions at a lower, but comparable,

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level to the strong CMV promoter. The TAP promoter shows great potential, as a useful method of controling the level of gene expression in porcine cells as well as a means to control transgenes with the host immune system. Of the few other cell types we tested in the current study, the TAP promoter demonstrated no promoter activity in porcine fibroblasts and granulose cells. Approximately 1.5% of the transfected PK-15 (porcine kidney epithelium originated) cells expressed a low level of GFP driven by the TAP promoter, the expression, however, did not increase in response to the LPS challenge. This data suggests that there may be a low level of the TAP promoter activity in nontracheal epithelial cells, although the activity level was minimal compared to that in tracheal epithelial cells (1.5% vs. 67.8%). Further in vivo study will help to confirm the specificity. The annual loss due to airborne bacterial diseases in the pork industry in North America is significant, resulting in increased days to market, cost of medication, and overall reduced income (Rohrbach et al., 1993). Production of infection-resistant pigs by a transgenic approach has become an attractive solution to this persistent problem. The ability of the TAP promoter to control gene expression in an inducible manner in the porcine respiratory tract presents an important application potential for transgenic study. By linking the TAP promoter to a gene sequence encoding an anti-bacterial protein, it may be possible to produce transgenic pigs that can express the anti-bacterial protein in an inducible manner in response to bacterial infection in the respiratory tract. The authors wish to thank the staff of Domingos Meat Packers, Department of Animal and Poultry Science Abattoir, University

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of Guelph, and the Conestoga Meat Packers for their assistance in collecting porcine tracheal samples.

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