ABSTRACT
To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities against Actinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection with A. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity against A. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance against A. pleuropneumoniae infection.
INTRODUCTION
The availability of antibiotics for treating bacterial infection has significantly improved the health of animals and humans. Administration of antibiotics at low doses to food animals has been practiced to promote better health and animal performance in many regions (1). To reduce the development of antimicrobial resistance, approaches to decrease antibiotic use in animal production are needed.
Antimicrobial peptides (AMPs), a family of short cationic amphiphilic peptides with antimicrobial and immune modulation activities, exist in nearly every life form as natural anti-infective therapeutic agents (2). Some AMP genes have been used to generate transgenic (TG) mice to enhance resistance to bacterial infection. For example, expression of a synthetic cecropin-class lytic peptide in TG mice displayed enhanced resistance to Brucella abortus (3). Expression of an additional porcine cathelicidin peptide, PR-39, in mice showed increased resistance to group A Streptococcus infection (4). Furthermore, enhanced resistance to Actinobacillus suis infection was observed in proptegrin-1 TG mice (5). These studies demonstrate that AMPs potentially can lead to the development of infection-resistant animals, at least in TG mouse models. However, few reports are available on large food animals such as pigs.
Defensins comprise a major family of AMPs, playing an important role in innate immunity. Porcine beta-defensin 2 (PBD-2) is thought to be an important AMP. PBD-2 provides a first line of defense against bacterial infection in pigs, because this AMP is highly expressed in epithelial cells (6, 7); moreover, PBD-2 demonstrates excellent antimicrobial activity with a broad spectrum but without hemolytic activity under physiological conditions (8). In the current study, we generated PBD-2-overexpressing pigs by somatic cell cloning and evaluated their resistance to bacterial infection by direct or cohabitation infection with Actinobacillus pleuropneumoniae, which is the causative agent of porcine pleuropneumonia, a highly contagious respiratory disease responsible for major economic losses in the swine industry worldwide. This disease is characterized by hemorrhagic, fibrinous, and necrotic lung lesions (9–11). In our study, PBD-2 was overexpressed in various tissues, such as the heart, liver, spleen, lungs, kidneys, and jejunum, of the TG pigs. The pigs showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. Our data suggest that overexpression of a native defensin is feasible to confer enhanced resistance to bacterial infection in pigs. This approach is promising in the development of disease-resistant animals, which can reduce the need for antibiotics in the animal industry.
MATERIALS AND METHODS
Construction of PBD-2-expressing plasmid and validation in PK-15 cells.The full-length (216 bp) cDNA of PBD-2 peptide was prepared from the liver of a healthy Large White pig by using reverse transcription-PCR (RT-PCR) with primers PBD2-01 (5′-CCG GAA TTC ATG TGG GCC CTC TGC TTG-3′) and PBD2-02 (5′-CCG CTC GAG TCA GGG TCA GCG GAT GCA-3′). The PCR product was cloned into the mammalian expression vector pCAGGS (Addgene, Cambridge, MA, USA) to generate pCAG-PBD2. The SspI-XhoI fragment, containing the CAG promoter (including the cytomegalovirus immediate-early [IE] enhancer, chicken beta-actin promoter, chicken beta-actin intron, and rabbit beta-globin intron) and porcine beta-defensin cDNA (PBD-2), was released from pCAG-PBD2 and then inserted between the same restriction sites as those of the mammalian expression vector pcDNA3.1(+) (Invitrogen, Shanghai, China). The resultant recombinant plasmid was termed pcCAG-PBD-2. pcCAG-PBD2 and empty vector pcDNA3.1(+) were transfected into porcine kidney (PK-15) cells by using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's instructions. To generate stable cell lines, the cells were selected with G418 (600 μg/ml) for 14 days, and single colonies were isolated. After expansion, the cells were selected. PBD-2 expression was confirmed by indirect immunofluorescence assay (IFA) with the PBD-2 monoclonal antibody 1013, prepared in our laboratory (12). The antibacterial activity of the recombinant PBD-2 peptides expressed in the PK-15 cells was determined by disk diffusion susceptibility test; in this procedure, the cells were frozen and thawed thrice, and 20 μl of the lysate and cell culture supernatant was added onto a 6-mm-diameter filter paper disk (1-mm thickness; Whatman, England). All of the disks were incubated with A. pleuropneumoniae 4074 on a Trypticase soy agar plate at 37°C overnight.
Generation of TG pigs.This research was conducted in accordance with the Chinese Animal Care Guidelines. All of the experiments were approved by the Animal Experimental Committee and the Transgenic Safety Administrative Committee of Huazhong Agriculture University. The PBD-2-expressing cassette was released from pcCAG-PBD2 with AccI, which contains the CAG promoter, PBD-2 cDNA, and bovine growth hormone (BGH) poly(A) signal. Fetal fibroblasts from a male Large White pig were transfected with the transgene cassette, and fibroblast cell lines stably overexpressing PBD-2 were established. The TG pigs were generated by BGI Ark Biotech Co., Ltd. (Shenzhen, China), via handmade a cloning method as described previously (13). DNA was isolated from ear snip samples of the offspring, and PCR was performed to identify the TG pigs by using primers NP03 (5′-GCT GGT TGT TGT GCT GTC TC-3′) and NP04 (5′-AGG TCC CTT CAA TCC TGT TG-3′) for the transgene cassette and GAPDH-01 (5′-CAA GGT CAT CCA TGA CAA CTT TG-3′) and GAPDH-02 (5′-GTC CAC CAC CCT GTT GCT GTA G-3′) for GAPDH as a housekeeping gene. Pig ear genomic DNA (10 μg) was digested with EcoRI overnight and subjected to Southern blot analysis for transgene integration status. PBD-2 cDNA was used as a probe. All of the procedures were run in accordance with the handbook of the DIG (digoxigenin) High Prime DNA labeling and detection starter kit I (Roche, Germany). Sperm was collected from male TG founders and crossed with wild-type (WT) Large White sows by artificial insemination to produce numerous TG and non-TG pigs for transgene expression and anti-infection studies.
RT-PCR.Total RNA was extracted from fetal porcine fibroblasts or pig tissues via the SV total RNA isolation system (Promega, Shanghai, China), digested with RNase-free DNase I, and subsequently converted into cDNA by using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Biochemical Product Co., Ltd., Beijing, China). PCR amplifications were performed using primers NP03 and NP04 for the PBD-2 transgene and primers GAPDH-01 and GAPDH-02 for the housekeeping gene GAPDH.
Competitive chemiluminescence enzyme immunoassay.Aliquots of tissues (approximately 400 mg) and PBD-2 TG pigs or their WT littermates were lysed with 1 ml of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Hangzhou, China). PBD-2 protein levels were evaluated by competitive chemiluminescence enzyme immunoassay (CLEIA) as described in our previous report (12).
Immunohistochemistry.Tissues were harvested and fixed in 10% neutral buffered formalin. Immunoreactivity for PBD-2 was visualized using the SABC-AP (mouse IgG) kit (Boster Biotech, Wuhan, China), and PBD-2 mouse monoclonal antibody 1013 was diluted 1:2,000 as the primary antibody.
ELISAs.The serum IgG titers to the toxin ApxIVA as well as to whole cells of A. pleuropneumoniae were measured before the bacterial infection studies. The ApxIVA-enzyme-linked immunosorbent assay (ELISA) kit was purchased from Wuhan Keqian BioTech (Wuhan, China). The negative and positive serum samples of this kit were used as negative and positive controls in the whole-cell ELISA. For whole-cell ELISA, 50 μl of fresh A. pleuropneumoniae (1 × 108 CFU/ml in phosphate-buffered saline [PBS]) was added into a 96-well ELISA plate. The plate was dried at 37°C, and 200 μl precooling methanol was added for 15 min at room temperature. The plate was washed three times with PBS-T (PBS with 0.1% Tween 20) and then blocked with 1% bovine serum albumin (BSA) in PBS-T for 2 h at 37°C. After washing, the serum samples of pigs and negative- and positive-control sera were diluted 1:50 in 1% BSA and then added to the plate. After incubating at 37°C for 1 h, the plate was washed thrice and goat anti-porcine IgG-horseradish peroxidase (HRP) (diluted 1:5,000; Southern Biotechnology Associates Inc., Birmingham, AL, USA) was added and incubated at 37°C for 1 h. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB; Vazyme, Nanjing, China) was added and incubated for 10 to 15 min at 37°C. Finally, 1 M H2SO4 was added to stop color development, and the optical density (OD) was measured at 450 nm.
Bacterial infection.A. pleuropneumoniae 4074 (serovar 1) was cultured on tryptic soy agar (TSA) or in tryptic soy broth (TSB; Difco Laboratories, USA) supplemented with 10 μg/ml of NAD and 10% (vol/vol) filtered cattle serum at 37°C as described previously (14). The susceptibility of the TG pigs to A. pleuropneumoniae infection was evaluated in two infection studies. In study 1 (direct infection), six 2-month-old TG pigs and four WT littermates were intratracheally infected with 1 × 106 CFU of A. pleuropneumoniae 4074. Two TG and two WT pigs were injected intratracheally with physiological saline as controls. The bacterial loads in the lungs were measured at 2 days postinfection (dpi) by the plate count method (15). All of the bacteria recovered from the lungs were identified by PCR using A. pleuropneumoniae-specific primers ApxIVA-1L and ApxIVA-1R (16).
In study 2 (cohabitation infection), one 2-month-old WT pig was intratracheally infected with 1 × 108 CFU of A. pleuropneumoniae 4074 and then housed with four 2-month-old TG pigs and four WT littermates. The pigs were monitored and scored using a scale of 0 to 4 to show the increased signs of the disease after cohabitation infection until 10 dpi, as described by Oldfield et al. (15). Afterward, all of the pigs were humanely euthanized. The bacterial loads in the lungs were measured and identified at 10 dpi as described above. The clinical score of lung was performed with the consolidation lung lesion score (LLS) (17).
Validation of the effect of additional PBD-2.Blood samples were collected by venous puncture of the ears from 2-month-old TG and WT pigs. The serum concentration of PBD-2 was evaluated by CLEIA as described above. Cells of A. pleuropneumoniae were collected and resuspended in physiological saline before being added to the heat-inactivated serum samples. Up to 1 ml of porcine serum was mixed with 100 μl of A. pleuropneumoniae cells (about 5 × 103 CFU), and the mixtures were incubated at 37°C. These mixtures were harvested after 1 h, and bacterial numbers were detected by the plate count method.
Statistical analysis.Student's t test was used for statistical analysis, and all of the data are expressed as means ± standard errors of the means (SEM). Differences in values were considered significant at P < 0.05.
RESULTS
Expression of PBD-2 confers antimicrobial activity to porcine kidney cells.To validate whether PBD-2 can be overexpressed in porcine cells, the recombinant plasmid pcCAG-PBD2 (Fig. 1A) was transfected into PK-15 cells. Stably transfected cell clones then were selected under G418 pressure. Overexpression of PBD-2 in the cytoplasm of the pcCAG-PBD2-transfected cells was verified by IFA (Fig. 1B). Culture supernatant of the pcCAG-PBD2-transfected cells showed obvious antibacterial effects by disk zone inhibition assay, whereas the cell lysates displayed the same killing activities against A. pleuropneumoniae as the mock-treated control. In contrast, no antibacterial activity was detected in the culture supernatant or cell lysates of the mock-treated cells (Fig. 1C).
Transfection of porcine beta-defensin 2 (PBD-2) gene to PK-15 cells confers bactericidal activity. (A) The recombinant vector pcCAG-PBD-2, containing the cytomegalovirus early enhancer region (IER), chicken β-actin promoter (CBAP), chicken β-actin intron (CBAI), rabbit β-globin intron (RBGI), PBD-2 gene, BGH pA, and neomycin resistance gene (Neo), was prepared for the construction of stable transfected PK-15 cells. (B) Indirect immunofluorescence analysis showed that the expression of PBD-2 peptides was higher in the stable transfected cells than that in mock-infected cells (empty vector). (C) Obvious inhibition zones also were found in the cell lysates of the stable transfected cells in the disk zone inhibition assay.
Production and characterization of PBD-2 transgenic pigs.To assess the antibacterial potential of PBD-2 in vivo, two male TG founder pigs (F0) overexpressing PBD-2 were generated by handmade nuclear transfer from the stable PBD-2-overexpressing fetal porcine fibroblasts. In these TG pigs, the PBD-2 gene was driven by the CAG promoter (Fig. 2A), which allowed expression in all cell types (18). The TG founders then were crossed with WT Large White sows to generate TG pigs (F1). PCR (Fig. 2B) and Southern blot analyses (Fig. 2C) were performed using the genomic DNA from the TG pigs and their WT littermates, and the results showed successful addition of the transgene.
Construct of transgenic (TG) pigs overexpressing porcine beta-defensin 2 (PBD-2). (A) Transgene cassette of PBD-2. Also shown are the confirmation of TG pigs using PCR (B) and Southern blot analysis (C).
Transgene expression was detected by RT-PCR in the tissues of the TG pigs raised from two TG founders (Fig. 3A). The CLEIA analysis revealed that PBD-2 was expressed at high levels in the liver, spleen, and lung, whereas PBD-2 in the jejunum was expressed at low levels in both TG and WT pigs (Fig. 3B). Moreover, the PBD-2 protein concentrations in the tissues of TG pigs were significantly higher than those of the WT littermates. The PBD-2 protein expression levels of TG pigs in the liver, spleen, and lung were approximately 8 ng/mg, and those in the jejunum were approximately 4 ng/mg, which were significantly higher than those of WT pigs. Immunohistochemistry detected the overexpression of PBD-2 peptide in most TG pig tissues by using PBD-2 monoclonal antibody. The immunohistochemical staining of lung showed stronger PBD-2 signals in the bronchioles and alveoli of the TG pigs than in their non-TG littermates (Fig. 3C).
Expression of inserted porcine beta-defensin 2 (PBD-2) gene in transgenic (TG) pigs. (A) The mRNA of the inserted PBD-2 gene was detected by specific primers in TG pigs but not in wild-type (WT) pigs. (B) ELISA of PBD-2 protein concentrations in tissue samples of TG pigs and their WT littermates. Data represent the means ± SEM (WT, n = 3; TG, n = 3). **, P < 0.01. (C) Lung sections from 3-month-old PBD-2 TG and WT pigs processed for immunohistochemical stain by using a mouse monoclonal antibody against PBD-2 (magnification, ×40).
Clinical characterization of TG pigs.Except for two founders (one non-TG and one TG founder) that showed lameness, all TG pigs and their non-TG littermates showed neither abnormal development nor abnormal behavior. No significant abnormal parameter was found in the routine blood test and clinical biochemical examination (Table 1).
Blood routine and biochemical examination
ELISAs.Before bacterial infection, all pigs were negative for ApxIVA antibody, indicating no A. pleuropneumoniae infection in these pigs (see Fig. S1A in the supplemental material). Moreover, the serum IgG titers to whole cells of A. pleuropneumoniae in the TG and WT pigs were not significantly different from those for the negative serum (see Fig. S1B). Thus, there are no maternal antibodies or cross-reactive antibodies toward A. pleuropneumoniae in these pigs before infection.
Resistance to intratracheal challenge with A. pleuropneumoniae.To determine whether PBD-2 overexpression enhanced the antibacterial resistance in vivo, we intratracheally inoculated 1 × 106 CFU of A. pleuropneumoniae strain 4074 into PBD-2 TG pigs and their WT littermates in study 1. Following challenge, all pigs were gasping and then humanely euthanized at 2 dpi. The lungs of TG pigs and their WT littermates were collected. PCR results showed that almost all of the colonies recovered from the lungs were A. pleuropneumoniae. Significantly fewer A. pleuropneumoniae cells were isolated from TG pigs than from WT pigs (P < 0.01) (Fig. 4A). These observations confirm that the overexpression of PBD-2 in TG pigs increased bacterial resistance.
Bacterial recovery from the lungs of challenged pigs. Quantitation and representative culture of A. pleuropneumoniae CFU recovered from transgenic (TG) or wild-type pigs used in study 1 (A) and study 2 (B). Data represent the means ± SEM.
Resistance to cohabitation infection by A. pleuropneumoniae.Although A. pleuropneumoniae is an obligate respiratory bacterium and infects pigs via airborne particles, intratracheal inoculation is not the natural route of infection of this bacterium. In study 2, to mimic the natural infection of A. pleuropneumoniae, we carried out a cohabitation infection experiment to further evaluate the antibacterial potential of PBD-2 TG pigs. Significantly fewer A. pleuropneumoniae organisms were isolated from TG pigs than from WT pigs in study 2 (P < 0.05) (Fig. 4B). Although no significant difference was found in the survival rate (Fig. 5A), the lung lesion scores of TG pigs were significantly lower than those of WT pigs (Fig. 5B), and improved clinical scores also were found in TG pigs, as shown in Fig. 5C. A directly infected WT pig died at 3 dpi. This pig was housed with WT and TG pigs. Three WT pigs showed obvious clinical symptoms in the first 2 days, whereas the TG pigs had no significant symptoms. At 3 and 5 dpi, two out of four WT pigs died because of infection. Subsequently, one TG pig died at 6 dpi. In the last days, the remaining two WT pigs showed severe clinical symptoms and had clinical presentations with the highest scores at 10 dpi. Three out of four TG pigs survived to the end of the experiment and had lower scores than the surviving WT pigs. All of the remaining pigs were humanely euthanized at 10 dpi, and their lungs were removed for analysis. Severe congestion, hemorrhages, and edema were apparent in the lungs of WT pigs, but only mild congestion and edema were found in those of TG pigs (Fig. 5D). Clinical signs were not observed in control pigs.
Overexpression of porcine beta-defensin 2 (PBD-2) enhanced resistance to cohabitation infection by A. pleuropneumoniae. In study 2, one WT pig infected with A. pleuropneumoniae at day 0 was housed with four WT and four TG “in-contact pigs.” (A) Survival curve. (B) Lung lesion score. (C) Mortality and bacterial shedding data from study 2. Numbers for each day are clinical scores. Pigs were scored 0 if no signs of disease were found, and pigs showing serious clinical symptoms were scored 4. Survivals are showed by the length of the gray bar, and the terminal block in black indicates the date of death. Two WT and three TG pigs survived up to day 10 postinfection and were killed and autopsied immediately. (D) Representative photographs of gross and microscopic injury of lungs. The lungs and tissue sections from pigs inoculated with physiological saline were normal and lack pathological findings. The lungs of WT pigs infected with A. pleuropneumoniae showed hemorrhages, edema, severe congestion, and neutrophilic infiltrates. The lungs of TG pigs infected with A. pleuropneumoniae showed milder hemorrhages, moderate congestion, and mild neutrophilic infiltrates (H&E, hematoxylin-eosin).
Heat-inactivated TG pig serum inhibits bacterial reproduction.The CLEIA analysis showed that the PBD-2 concentration in the serum of TG pigs was significantly higher than that in the serum of WT pigs (Fig. 6A). To investigate the antibacterial activity of the PBD-2 peptide generated in the serum of TG pigs, we incubated heat-inactivated serum samples with A. pleuropneumoniae. Figure 6B shows that the bacterial number in the serum of WT pigs was significantly higher than that in TG pigs after 1 h of incubation (P < 0.01). These results indicate that the additional PBD-2 was active and PBD-2 overexpression could effectively inhibit the bacterial reproduction in serum.
Additional PBD-2 in the serum of transgenic (TG) pigs inhibited the growth of bacteria. (A) The concentrations of PBD-2 in the serum samples of TG pigs were significantly higher than in those of wild-type (WT) pigs. (B) Lower bacterial numbers were found in heat-inactivated serum samples from TG pigs. Data are the means ± SEM. WT, n = 3; TG, n = 3. **, P < 0.01.
DISCUSSION
Several studies have demonstrated that increased defensin expression in mice can increase resistance to bacterial infections (4, 5, 19). PBD-2 possesses high antimicrobial activities against a broad range of microorganisms. Moreover, it generates limited hemolytic activity against pig red blood cells (8). Recent studies have shown that the PBD-2 gene expresses mainly in epithelial cells (20). Meishan pigs have good disease resistance and high levels of PBD-2 in tongue, oral mucosa, and lung (7, 21). Therefore, this defensin may contribute to resistance to bacterial infection. We determined whether the overexpression of a porcine defensin gene could decrease susceptibility to bacterial infection in pigs. The results showed that the PBD-2 gene inserted into the genome of pigs produced high levels of antimicrobial peptide in TG tissues and enhanced the resistance to A. pleuropneumoniae infection in TG pigs. These findings demonstrate that overexpression of PBD-2 is promising in developing a disease-resistant animal.
In this work, a PBD-2 expression vector, pcCAG-PBD-2, was constructed in which BPD-2 expression was driven by the CAG promoter. This promoter is used to constructively express Kaede, a photoconvertible fluorescence protein in TG mice (22). An in vitro transfection study demonstrated that this promoter also could drive PBD-2 overexpression in porcine cells. Interestingly, the cell culture supernatant showed higher antibacterial activity than the cell lysate of the stably transfected PK-15 cells. This result indicates that posttranslational processing is important for the antimicrobial activity of peptides. The inactive form of peptides may have lower or even no antimicrobial activities compared with that of the mature peptides secreted to the supernatant. After validating the transgene construct in the porcine cells, TG pigs were generated by a handmade cloning method as described previously (13). The TG pigs were fertile and phenotypically normal and showed no significant abnormality in routine blood tests and blood biochemical examinations. The PBD-2 concentration in the serum of TG pigs was approximately 0.3 μg/ml, which was 100-fold higher than that of the WT pigs. Synthetic mature PBD-2 peptide with a concentration of 16 to 32 μg/ml has been reported to kill the most susceptible bacteria within 3 h (8). Although this concentration is much higher than that in the serum of TG pigs, heat-inactivated serum samples isolated from TG pigs showed a significantly inhibiting effect on bacterial reproduction.
We infected the TG pigs with A. pleuropneumoniae using two in vivo methods. In study 1, the pulmonary clearance of bacteria after direct infection through intratracheal injection in TG and WT pigs was compared. The TG pigs showed significantly lower bacterial burden in the lungs compared to that of the WT pigs. This finding is similar to those of previous reports, which demonstrated that the expression of an additional antimicrobial peptide can enhance the killing rate of bacteria in mice (4, 5). Study 2 was designed to investigate whether the results obtained in study 1 showing reduced bacterial burden in the TG-challenged lungs minimized susceptibility to infection via contact exposure to infected pigs, which was more similar to the natural infection of this disease. Although there is no significant difference between the survival rate of TG and WT pigs, the improved clinical scores, significantly reduced lung lesion scores, and pulmonary bacterial burden suggested that overexpression of PBD-2 enhanced resistance of TG pigs to A. pleuropneumoniae.
Although the results obtained in this study suggested that enhancing bacterial clearance in the presence of PBD-2 contributed to the increased resistance to A. pleuropneumoniae infection observed in the PBD-2 TG pigs, other explanations for this phenotype still are possible. Defensins are multifunctional peptides and are speculated to be important components of immunity (23). Defensins possess immunosuppressive activity, proinflammatory effects, and chemoattractant effects (24–26), although their precise in vivo role has not been clarified (25). Therefore, overexpression of PBD-2 may increase the release of chemokines, cytokines, or other immune factors to help resistance to infection.
In summary, this study demonstrated that the TG pigs overexpressing PBD-2 displayed enhanced resistance to A. pleuropneumoniae infection. This work provides a good foundation for the development of a disease-resistant animal which could reduce the use of antibiotics in animal industry.
ACKNOWLEDGMENTS
This work was supported by the National Transgenic Breeding Projects (2009ZX08006-006B), National Basic Research Program of China (973 program, 2012CB518802), and Special Fund for Agro-scientific Research in the Public Interest (201303034-11).
We are grateful to Yutao Du from BGI Ark Biotech Co., Ltd. (Shenzhen, China), for technical support in the handmade cloning of transgenic pigs.
We have no conflicts of interest to declare.
FOOTNOTES
- Received 22 December 2014.
- Returned for modification 19 January 2015.
- Accepted 24 April 2015.
- Accepted manuscript posted online 27 April 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.03101-14.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
