Deposition of bioactive human epidermal growth ... - The FASEB Journal

The FASEB Journal article fj.14-264739. Published online February 17, 2015.

The FASEB Journal • Research Communication

Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter Tae Sub Park,* Hyo Gun Lee,† Jong Kook Moon,‡ Hong Jo Lee,† Jong Won Yoon,† Bit Na Rae Yun,‡ Sang-Chul Kang,‡ Jiho Kim,‡ Hyunil Kim,‡ Jae Yong Han,†,1 and Beom Ku Han‡,1 *Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Korea; †Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea; and ‡Optipharm, Osongsaengmyeong 6-ro, Cheongju-si, Chungcheongbku-do, Korea Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene-encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen’s oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codon-optimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 59-UTR. Subsequently, a 39-UTR carrying the poly-A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW-derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg-derived EGF afforded slightly higher levels of re-epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct-specific minisynthetic promoter.—Park, T. S., Lee, H. G., Moon, J. K., Lee, H. J., Yoon, J. W., Yun, B. N. R., Kang, S.–C., Kim, J., Kim, H., Han, J. Y., Han, B. K. Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an

ABSTRACT

Abbreviations: 23ERE, 2 estrogen response elements; ALV, avian leukosis virus; BLAST, Basic Local Alignment Search Tool; CEF, chicken embryonic fibroblast; cEGF, codonoptimized human epidermal growth factor; CMV, cytomegalovirus; COUP, chicken ovalbumin gene upstream promoter; CRISPR, clustered, regularly interspaced, short palindromic (continued on next page)

0892-6638/15/0029-0001 © FASEB

oviduct-specific minisynthetic promoter. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: bioreactor • piggyBac transposon • primordial germ cell • transgenic chicken IN THE 1980S, GENE DELIVERY technologies were used to generate transgenic animals carrying transgenes integrated into host genomes (1). Such genetically engineered animals created considerable excitement and opened new eras in many fields, including basic research, disease modeling, and cancer biology. Also, industrial applications in agriculture and medicine were increasingly developed. Transgenic technology yields comprehensive information on the functions and regulatory mechanisms of novel genes and allows cancer development and differentiation to be explored by tracking target gene products in vivo (2). The first transgenic mouse was produced by microinjection of purified plasmid DNA into the pronuclei of fertilized embryos (3), and Palmiter et al. (4) generated transgenic offspring via germline modification of a transmitted transgene. In 1983, “Mighty Mice” featuring integration of the human growth hormone gene fused to the mouse methalliothionein-1 gene promoter exhibited dramatic growth (5). The potential applications of transgenic technology include emerging biotechnologic approaches to the production, propagation, and management of desirable (economically important) characteristics of livestock. Transgenic birds have many practical and industrial uses, serving as disease models and animal bioreactors (2). Recently, transgenic chicken production systems have become well established, using both nonviral transposonmediated gene transfer and viral induction (6–11). The 1 Correspondence: J.Y.H., Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. E-mail: [email protected]. kr; B.K.H., Optipharm, Osongsaengmyeong 6-ro, Cheongju-si, Chungcheongbku-do, 363-954, Korea. E-mail: bkhan@optipharm. co.kr doi: 10.1096/fj.14-264739 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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first report on production of transgenic chickens used the microinjection technology developed for mammals (12). However, both technical difficulties and bird mortality have rendered this approach little used today for avian transgenesis. A most promising strategy for the delivery of foreign transgenes involves virus-mediated infection of host cells and tissues. Transgenic chickens have been produced via injection of recombinant avian leukosis viruses (ALVs) into the blastoderm of unincubated fertilized eggs (13). This technique has been valuable when used to insert foreign genes into the chicken germline. Viral infection has also been used to produce transgenic quails and songbirds (14, 15). Particularly, modification of target genes in zebra finches (which are widely used to study vocal learning) yields crucial clues aiding the understanding of the molecular processes involved in such learning (15). To generate transgenic birds, any transgene must be transmitted to the next generation via germ cells. Thus, manipulation of germ cells including primordial germ cells (PGCs), which serve as precursor cells at early developmental stages, has been attempted in efforts to improve the efficacy of transgenesis in avian species (9–11). After transfer of virus-infected PGCs into recipient embryos, transgenic quails (9) or chicks (10, 11) carrying transgenes were identified in donor germ cell-derived offspring. This method is feasible because avian PGCs migrate through blood vessels to become localized in developing embryonic genital ridges. Thus, such cells can be isolated and reintroduced into blood vessels of recipient embryos. However, the numbers of PGCs that can be retrieved from the germinal crescent, blood vessels, or embryonic gonads at different developmental stages are rather restricted, and the retrieval procedures are time consuming and laborious. Recently, long-term culture systems for chicken PGCs have been established, and such cells exhibited higher germline-competent capacities (16, 17). In our previous report (8), we showed that germline transmission efficiency ranged from 90.4 to 98.9% when the SNUhp26 male PGC line was used. The in vitro culture technique can easily be expanded to produce high numbers of chicken PGCs, allowing stable transfection and selection of transgenes integrated into the genome. Using advanced culture systems for chicken PGCs, nonviral transgene delivery methods employing the piggyBac and Tol2 transposon elements have been developed (7, 8). Because virus use in transgenic animals raises biosafety issues, the development of nonviral strategies has been essential to allow industrial applications to proceed. Additionally, advantages of transposon use are efficient transposition into the genome, multi-insertion associated with nondisruption of functional genes (via integrating (continued from previous page) repeat; EGF, epidermal growth factor; ERE, estrogen response element; EW, egg white; FBS, fetal bovine serum; GFP, green fluorescent protein; H&E, hematoxylin and eosin; hEGF, human epidermal growth factor; IRES, internal ribosome entry site; KO, Korean Oge; MT, Masson’s trichrome; NRE, negative regulatory element; OV, ovalbumin; PBTR, piggyBac terminal repeats; PGC, primordial germ cell; rhIL1RN, recombinant human IL-1 receptor antagonist; SDRE, steroiddependent regulatory element; TALEN, transcription activator-like effector nuclease; WL, White Leghorn

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into introns and intergenic regions), and stable integration of large transgenes (18, 19). Furthermore, PGC culture systems may be used for gene targeting via homologous recombination (20) and for precision genomic editing of the chicken genome using state-of-the-art technologies such as those involving the transcription activator-like effector nuclease (TALEN) (21) and (programmable) clustered, regularly interspaced, short palindromic repeat [(CRISPR)-associated] technology. Ovalbumin (OV) is a major protein of eggs; ;2 g is found in a single hen’s egg. TALEN- or CRISPR-targeted insertion of genes of interest into the OV gene could afford very high-level production of bioactive biomaterials. In the present study, we generated transgenic chickens expressing human epidermal growth factor (hEGF) driven by a minisynthetic promoter allowing oviduct-specific expression, partially purified hEGF from transgenic hen egg white (EW), and analyzed the biofunctional activities thereof using the (in vivo) miniature pig model. Our results suggest that the use of transgenic hen eggs as a live bioreactor could be useful and practical for the high-level production of biomaterials required by the agricultural and biotechnologic industries. MATERIALS AND METHODS Experimental animal care The care and experimental use of chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University (permit number SNU-070823-5). All experimental chickens, thus White Leghorn (WL) and Korean Oge (KO) chickens, were maintained in accordance with a standard management program in operation at the University Animal Farm, Seoul National University, Korea. All procedures for animal management, reproduction, and embryo manipulation were governed by standard operating protocols. Codon optimization of the hEGF gene and construction of an expression vector Codons of the hEGF gene were optimized for expression in the hen using the Gallus gallus codon usage database (http://www. kazusa.or.jp/codon). A codon-optimized human epidermal growth factor (cEGF) gene was synthesized by Bioneer Company (Daejeon, Korea). There were 2 consecutive “chickenized” epidermal growth factor (EGF) genes synthesized; both contained internal ribosome entry site (IRES) sequences (Fig. 1A). To allow secretion of hEGF from the cells, a chicken lysozyme signal peptide sequence was placed before each hEGF gene. The 647 bp estrogen response element (ERE) of the chicken OV gene was cloned via genomic PCR using specific primers: forward 59-tgc aga aag atg cca ggt gg-39 and reverse 59-tct aga gag agt aag caa caa tct tct-39, and 2 EREs (23ERE) were next consecutively ligated. Subsequently, the 23ERE cassette was inserted between the 59-piggyBac terminal repeats (PBTR) and the 39-PBTR of the piggyBac transposon. A 3.0 kb segment including the chicken OV promoter (1.4 kb) with the 59-UTR (1.6 kb), and 1.6 kb of the 39UTR, including the OV poly-A tail sequences, was cloned by genomic PCR. After ligation of the synthesized cEGF between the 3.0 kb OV promoter and the 1.6 kb 39-UTR, the construct was ligated into the piggyBac transposon vector together with the 23ERE cassette (23ERE-OVcEGF). To explore the regulatory effect of the 23ERE sequence, a cEGF expression vector driven by the 3.0 kb OV promoter, and containing the 1.6 kb 39-UTR but without the

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PARK ET AL.

Figure 1. Codon optimization of the hEGF gene and the expression vectors constructed. A) The codons of the hEGF gene were optimized to reflect G. gallus codon usage (cEGF). The hEGF sequence and the optimized codons are indicated in blue and yellow, respectively. The lysozyme signal peptide sequence is indicated in red. There were 2 consecutive cEGF genes synthesized, both with IRES sequences. B) The 23ERE-OVcEGF expression vector contains 23ERE. The GFP-OVcEGF vector carries the GFP reporter gene driven by the CMV promoter, but no ERE sequence. 23ERE cassette, was also constructed, and the cytomegalovirus (CMV) promoter-green fluorescent protein (GFP) reporter (GFPOVcEGF) gene ligated therein, to allow us to trace transplanted donor PGCs in recipient embryonic gonads (Fig. 1B). Because the 39-UTR contains OV poly-A tail sequences, we did not add extra poly-A sequences to the 2 expression vectors (23ERE-OVcEGF and GFP-OVcEGF). For transfection of DT40 cells and chicken embryonic fibroblasts (CEFs), we constructed a cEGF expression vector carrying the CMV immediate-early enhancer/promoter and ligated this into the piggyBac transposon.

BIOACTIVE HEGF PRODUCTION IN TRANSGENIC HEN EGGS

PGC and chicken cell cultures for transgene transfection and selection A SNUhp26 male PGC line was established using d 6 (stage 28) WL embryonic gonads in our previous report (8) and was maintained in knockout DMEM (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 20% (v/v) fetal bovine serum (FBS) (Invitrogen, Life Technologies), 2% (v/v) chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 13 nucleoside mix (EMD Millipore, Temecula, CA, USA), 2 mM L-glutamine,

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13 nonessential amino acid mix, 2-ME, 10 mM sodium pyruvate, and 13 antibiotic-antimycotic mix (Invitrogen, Life Technologies). Human basic fibroblast growth factor (10 ng/ml; Koma Biotech, Seoul, Korea) was used to stimulate PGC proliferation. SNUhp26 PGCs were cultured at 37°C under an atmosphere of 5% (v/v) CO2 and at 60–70% relative humidity. Cultured PGCs were subcultured onto mitomycin-inactivated layers of mouse embryonic fibroblasts at 5–6 day intervals via gentle pipetting (in the absence of any enzyme treatment). All transfection and selection procedures followed those established in our previous report (8). Each cEGF expression vector, and CAGG-PBase (pCyL43), was cotransfected into the SNUhp26 PGC line via lipofection using the Lipofectamine reagent (Invitrogen, Life Technologies). At 1 day after transfection, G418 (to 300 mg/ml) was added to culture media to allow selection of both 23ERE-OVcEGF- and GFP-OVcEGF-expressing transductants, and GFP-expressing cells or colonies bearing the GFP-OVcEGF expression vector were observed via fluorescence microscopy during the course of G418-based selection. DT40 and CEF cells were maintained and subpassaged in DMEM supplemented with 10% (v/v) FBS and 13 antibioticantimycotic mixture (Invitrogen, Life Technologies). Chicken cells were cultured at 37°C under an atmosphere of 5% (v/v) CO2 and at 60–70% relative humidity. To generate cEGF-expressing DT40 and CEF sublines, CMV-cEGF plasmid DNA carrying a neomycin-resistance (NeoR) gene driven by the simian vacuolating virus 40 promoter was transfected into chicken DT40 cells using Lipofectamine, according to the manufacturer’s protocol. At 1 day after transfection, 300 mg/ml G418 was added to culture media, and selection was complete 2 weeks later. Microinjection and screening of transplanted donor germ cells in recipient embryos To inject selected transfected PGCs into recipient embryos, a small window was made on the pointed end of each recipient KO egg, and a 2 ml aliquot containing at least 3000 PGCs was microinjected via a micropipette into the dorsal aorta of the recipient embryo. Each egg window was sealed with paraffin film, and the eggs were incubated with the pointed end down prior to further screening and eventual hatching. Following such transfer, GFP-expressing PGCs (GFP-OVcEGF) were sought in the testes of recipients after 6 or 12 days of incubation. Recipient embryonic gonads were dissected at different stages, and live images of GFPpositive transplanted PGCs were obtained using a fluorescence or confocal laser-scanning microscope (LSM 700; Carl Zeiss, Wetzlar, Germany). Testcrossing and detection of transgenic chickens WLs with a dominant pigmentation inhibitor gene (I/I) and black KOs with a recessive pigmentation inhibitor gene (i/i) were used to obtain donor PGCs and recipient embryos, respectively. Via testcrossing analysis by mating with KO females (i/i), putative germline chimeras were identified by their offspring. Sperm from KO recipient male chickens (i/i) can father only black KOs because of the presence of only the recessive pigmentation inhibitor gene (i/i), whereas WL donor-derived sperm (I/I) can father white hybrids with the I/i genotype pigmentation genotype. GFP-expressing transgenic chickens were easily identified using a fluorescent excitation lamp fitted with appropriate filters (BLS Limited, Budapest, Hungary). Identification of the sites of integrated DNA in transgenic chickens Transgene insertion sites were identified using a DNA Walking SpeedUp Premix Kit-II (Seegene, Seoul, Korea) according to the

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manufacturer’s protocol. The PCR products after the third round of DNA walking were excised from the agarose gel, purified using a Power Gel Extraction Kit (Promega, Madison, WI, USA), and next cloned into the pGEM-T Easy Vector (Promega). The cloned PCR products were sequenced using an ABI Prism 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of the 59-flanking regions were analyzed using the Basic Local Alignment Search Tool (BLAST) Assembled Genome database (http://blast.ncbi.nlm.nih.gov/BLAST.cgi) and the UCSC Genome Bioinformatics’ browser (http://www.genome. ucsc.edu) to identify transgene insertion sites in the genomes of transgenic chickens. Transgene genotyping To generate homozygous chickens with 2 copies of the 23EREOVcEGF transgene at the same locus, #4767 (G1) hen was with transgenic males (G1) with the 23ERE-OVcEGF transgene at the same locus of chromosome 6. We used specific primer sets to distinguish between the wild-type and transgenic loci. In the presence of the transgenic locus, the forward primer (ERE F2; 59tgt gcc aag ttc tta tat cct ctg-39) binds to the chicken genome, but the reverse primer (ERE R2; 59-tcc taa atg cac agc gac gga-39) binds to the transgene. In detection of the wild-type locus, the primer set (ERE F1; 59-gac aga aat gag ccc ttc gg-39 ERE R1; 59-gct ttt gat ggg tct ggc at-39) binds to chicken genomic sequences at the locus of the transgene. Thus, a PCR product of 342 bp was derived from nontransgenic wild-type chickens, whereas a PCR product 468 bp in length was amplified from homozygous transgenic chickens. In heterozygous transgenic chickens, both the wild-type and transgenic loci were amplified, yielding 2 PCR amplicons of 342 and 468 bp, respectively. PCR was performed via initial incubation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 58°C (ERE F1/R1 for the wild-type locus) or 60°C (ERE F2/R2 for the transgenic locus) for 30 seconds, and 72°C for 30 seconds. All reactions were terminated by final incubation at 72°C for 7 minutes. Detection and purification of cEGF in the EW of transgenic hens The concentrations of cEGF in transgenic hen EW were determined using an hEGF ELISA kit (Koma Biotech) according to the manufacturer’s protocol. The antibody did not show any cross-reactivity with mouse EGF, hEGF receptor, and heparinbinding EGF. For the in vitro assay, cEGF in EW was partially purified via ammonium sulfate precipitation, and the wild hen EW was used as a baseline negative control. Thus, ammonium sulfate was added to 50% (w/v) to yolk-deprived EW, followed by mixing for 1 hour at 4°C. After centrifugation at 3000 3 g for 30 minutes, the supernatant was collected, and ammonium sulfate again was added to 50% (w/v), followed by stirring for 1 hour at 4°C. The final precipitate after centrifugation at 3000 3 g for 30 minutes was resuspended in an equal volume of PBS.

In vitro proliferation assay of EW-derived hEGF The in vitro proliferation assay of EW-derived hEGF employed human cervical cancer HeLa cells and the 3-(4, 5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay. After 3 days of treatment, optical density values were measured by ELISA, at 680 nm. First, we determined the effects of 0.01, 0.1, 1, and 1 ng/ml commercial hEGF (EMD Millipore) on HeLa cells. To explore the bioactivity of EW-derived hEGF, HeLa cells were exposed to EW only, EW-derived hEGF, and commercial hEGF and, 1 day


PARK ET AL.

later, were added to 96-well plates supplemented with DMEM with 10% (v/v) FBS. The hEGF concentration was adjusted to 1 ng/ml, informed by results of preliminary testing. All experiments featured 3 replicates of each treatment. In vivo wound repair assay of the bioactivity of cEGF from transgenic hen EW One-year-old miniature pigs (weight, 43–57 kg) were used in the in vivo wound assay. The pigs were anesthetized, and the back hair was shaved using an electrical clipper, followed by washing with 70% (v/v) alcohol. Circular wounds 25 mm2 in area and 8 mm deep were generated in the skin and left undressed. Animals were randomly assigned to 2 groups: EW-derived cEGF-treated and nontreated controls. Microscopic data on inflammation, fibroplasia, neovascularization, re-epithelialization, and collagen synthesis were obtained on day 3, 7, and 14. Criteria of histopathologic analysis for wound healing Processes of wound healing are complicated and dynamic responses in the body and generally divide into the inflammatory phase, proliferative phase, and maturation phase. Inflammatory phase as the first step of wound healing represents an acute inflammation to eliminate the wound tissue and derivatives by neutrophils. In fibroplasia as a proliferative phase, fibroblasts migrate and proliferate onto the wound tissue. Fibroplasia results in the process of extracellular matrix formation. Neovascularization is the formation process of a new network of blood vessels in the granulation tissues. In re-epithelialization of the epidermis, epithelial cells proliferate and resurface the wound. During the maturation phase, collagen forms tight cross-links to other collagen and with other protein molecules, increasing the tensile strength of the wound tissue. According to the criteria of histopathologic analysis for wound healing, hematoxylin and eosin (H&E)-stained sections were scored as grade 0 (2, absent), 1 (+, scant or mild), 2 (++, moderate), or 3(+++, abundant or severe) for epidermal remodeling. All slides were examined in a blinded manner by a surgical pathologist. Preparation of wounded skin and histopathologic analysis There were 4 animals from each group analyzed on day 3, 7, and 14 postwounding. Wound tissues were cut into small pieces and fixed in formalin [10% (v/v) formaldehyde in PBS] for histologic examination. Paraffin-embedded tissue blocks were obtained, sections (4 mm thick) from the midportions of wounds were stained with H&E after deparaffinization, and the collagen deposition was evaluated after double staining with H&E and Masson’s trichrome (MT). Under a microscope (Olympus BX53; Tokyo, Japan), each slide was given a histologic score of 0, 1, 2, or 3, associated with no remarkable lesion, or a mild, moderate, or severe lesion, respectively.

RESULTS

DT40 and CEFs, indicating that the codon-optimized EGF was constantly produced by and secreted from chicken cells (Supplemental Fig. S1). Screening of transplanted donor germ cells in recipient embryos After lipofection of 23ERE-OVcEGF or GFP-OVcEGF into the SNUhp26 PGC line, stably GFP-expressing PGCs were selected using G418. Upon transfection with GFP-OVcEGF, constant and strong expression of GFP was observed in most PGCs (Fig. 2A). Subsequently, GFP-expressing PGCs with the GFP-OVcEGF transgene were transplanted into recipient embryonic blood vessels to explore the migration capacities of such PGCs. GFPexpressing PGCs were detected in embryonic gonads of recipients 6 days later, indicating that the transferred PGCs had migrated and settled in the gonads (Fig. 2B). At 12 days (the time at which germ cell differentiation commences), GFP-positive germ cells had colonized the seminiferous tubule of the male testis (Fig. 2B). GFPpositive transplanted PGCs and endogenous GFP-negative PGCs were admixed in the seminiferous tubules and were in mutual contact (Fig. 2B). Testcrossing and detection of transgenic chickens After sexual maturation, putative germline chimeras were testcrossed by mating with KO female chickens to generate transgenic chickens derived from transplanted donor PGCs. All 5 donor PGC-transplanted roosters (3 for GFPOVcEGF and 2 for 23ERE-OVcEGF) produced white hybrid progeny derived from donor PGCs (Fig. 2C). The efficiencies of germline transmission of donor PGCs to offspring were 98.1 and 89.5% for the GFP-OVcEGF and 23ERE-OVcEGF transgene, respectively (Table 1). A total of 153 of the 166 chicks that hatched from the 5 male founders were donor PGC-derived hybrids, and the average rate of germline transmission was 92.2%. Among donor PGC-derived progenies, GFP-OVcEGF transgenic chicks could be easily identified under a fluorescent excitation lamp fitted with appropriate detection filters because GFP expression was strong in all transgenic chicks (Fig. 2C). Surprisingly, 92.3% (47 out of 51) of donorderived hybrids were GFP-expressing transgenic chicks (Table 1). In work with the 23ERE-OVcEGF expression vector, approximately half of donor-derived offspring (48 out of 102, 47.1%) were identified as transgenic upon genomic PCR analysis (Fig. 2D and Table 1). The germline transmission efficiency of GFP-OVcEGF was slightly higher than that of 23ERE-OVcEGF (98.1 vs.89.5%), but the efficiency of transgenic chick production using GFPOVcEGF was almost twice that of the 23ERE-OVcEGF transgene (92.3 vs. 47.1%) (Table 1).

Detection and purification of cEGF in DT40 and CEFs To explore the expression levels of chickenized hEGF (cEGF), an expression vector bearing the ubiquitous CMV promoter was transfected into the DT40 cell line and CEFs, and transformants were selected with G418 for up to 2 weeks. ELISA detected cEGF in the culture media of both BIOACTIVE HEGF PRODUCTION IN TRANSGENIC HEN EGGS

Identification of the sites of genomic integration in transgenic chickens Transgene insertion sites in the chicken genome were determined via DNA walking. There were 2 chickens transgenic for GFP-OVcEGF (#9716 and #9793) and 3 5

Figure 2. Migration analysis of GFP-expressing PGCs and detection of transgenic chicks. A) GFP-expressing chicken PGCs (SNUhp26 line) after transfection and selection (the left panel is bright field, and the right is the fluorescent field). B) Detection of GFP-expressing donor PGCs after transfer into recipient embryos. GFP-expressing PGCs (GFP-OVcEGF) were detected in recipient testes after 6 or 12 days of incubation. Recipient embryonic gonads were dissected, and live images of GFP-positive transplanted PGCs were obtained using a fluorescence microscope on day 6 and a confocal laser-scanning microscope on day 12. Arrowheads indicate transferred GFP-expressing PGCs and arrows endogenous GFP-negative PGCs. C) Transplanted donor germ cell-derived chicks and GFP-expressing transgenic chicks. The KO (i/i) recipient sperm fathered black KO (i/i) chicks, whereas WL (I/I) donor-derived sperm fathered white hybrids with the I/i pigmentation genotype. GFP-expressing transgenic chicks were identified using a fluorescent excitation lamp fitted with appropriate filters. D) Screening of cEGF transgenic (TG) chicks via genomic PCR analysis.

transgenic for 23ERE-OVcEGF (#4625, #4617, and #4767) analyzed (Fig. 3). Apart from #4625 and #4617 (both with transgenes in chromosome 6), the transgenes were inserted into different chromosomal regions (Fig. 3 and Supplemental Fig. S2). All sites of integration exhibited unique TTAA sequences between the chicken genome and the transgene; this is the footprint of the piggyBac transposon and associated transposase activity (Fig. 3 and Supplemental Fig. S2). Additionally, all transgenes were inserted into intergenic regions and, thus, did not disrupt functional genes (Table 2).

Genotyping of transgene-bearing chickens We finally generated a homozygous transgenic line (G2) carrying 2 copies of the 23ERE-OVcEGF transgene on chromosome 6. To explore the genotype thereof, we designed 2 primer sets amplifying the wild-type or transgene locus (Fig. 4A). A PCR band of 342 bp was produced only by nontransgenic wild-type chickens, whereas a band of 468 bp was specifically amplified only from homozygous transgenic chickens (Fig. 4B). In heterozygous transgenic chickens with both wild-type and transgenic loci, both

TABLE 1. Production of transplanted donor germ cell-derived chicks and transgenic chicks from germline chimeras via testcross analysis

Expression vectors

GFP-OVcEGF

23ERE-OVcEGF

Germline chimera ID

A1812 A1815 A1816 Subtotal 9056 9058 Subtotal

Number of hatched chicks

21 12 19 52 34 80 114

Number of donor-derived chicks (%)a

21 12 18 51 27 75 102

(100.0) (100.0) (94.7) (98.1) (79.4) (93.8) (89.5)

Number of transgenic chicks (%)b

21 10 16 47 20 28 48

(100.0) (83.3) (88.9) (92.3) (74.1) (37.3) (47.1)

a The percentage of donor-derived chicks among the total number of hatched chicks. bThe percentage of transgenic chicks among the total number of donor germ cell-derived chicks. ID, identification.

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Figure 3. Identification of transgene insertion sites in the genomes of transgenic chickens via DNA walking. The transgene locus map of transgenic chicken #4767, in which the transgene was localized to chromosome 6 (Chr.), is shown. seq., sequence.

PCR amplicons (342 and 468 bp) were amplified. We thus proceeded to compare cEGF expression levels in EWs of heterozygous and homozygous transgenic lines. Detection and in vivo bioactivity assay of cEGF from transgenic hen EW The EW cEGF expression levels in G1 chickens carrying the 23ERE-OVcEGF transgene ranged from 0 to .10 ng/ml; we could not detect cEGF in G1 transgenic hens carrying GFP-OVcEGF (Fig. 5A). In addition, the 23ERE-OV promoter cassette did not show any expression leakage of the transgene in the blood of transgenic chickens (data not shown). We next generated homozygous transgenic TABLE 2. Identification of the integration loci in transgenic chickens Vectors

GFP-OVcEGF 23ERE-OVcEGF

Transgenic ID

Chromosome

#9716 #9793 #4625 #4617 #4767

4 19 21 21 6

Integration site

Intergenic Intergenic Intergenic Intergenic Intergenic

The integration loci of 5 transgenic chickens were determined to be in intergenic regions, thus without disruptions of endogenous functional genes. ID, identification.


chickens by mating the female #4767 and male #4630, which exhibited the same integration locus on chromosome 6. In homozygous hens, the cEGF expression levels were higher than those of G1 chickens, attaining 20 ng/ml (Fig. 5B). After partial purification, the yield and fold purification of cEGF from transgenic eggs were 50% and 10-fold, respectively. We obtained 60 ng/ml cEGF solutions via concentration after purification. In vitro assay of the proliferative capacity of EWderived hEGF First, we determined the commercial hEGF concentration effective to trigger proliferation of HeLa cells; this was 1 ng/ml (Supplemental Fig. S3). To explore the bioactivity of EW-derived hEGF, we performed a proliferation assay using 3 materials: EW alone, EW-derived hEGF (1 ng/ml), and commercial hEGF (1 ng/ml). EW-derived hEGF exerted a proliferative effect similar to that of commercial hEGF (Fig. 5C). Both EW-derived and commercial hEGF significantly enhanced HeLa cell proliferation, compared to EW alone (Fig. 5C; P , 0.05). Next, the in vivo bioactivity of cEGF purified from EW of transgenic eggs was evaluated using a miniature pig wound skin model. On d 3 post-treatment, severe inflammation was observed in both nontreated and treated lesions, which exhibited major populations of neutrophils. From day 7

Figure 4. Transgene genotyping in G2 transgenic chickens. A) The specific primer sets used to detect wild-type and transgenic loci. B) A 342 bp PCR product was produced by nontransgenic wild-type chicken DNA, whereas a 468 bp PCR product was amplified from homozygous (homo) transgenic chickens. In heterozygous (hetero) transgenic chickens, both the wild-type and transgenic loci were amplified, and both PCR amplicons were observed.

7 to day 14 post-treatment, tissue regeneration exemplified by fibroplasia, neovascularization, re-epithelialization, and collagen synthesis was evaluated (Fig. 5D). The inflammation level did not differ between the 2 groups. However, fibroplasia and neovascularization scores in the EW-derived cEGF-treated group were slightly higher than those of the nontreated group (Fig. 5D and Table 3). Thus, EW-derived cEGF enhanced tissue regeneration of wounded skin. In contrast, inflammation was continuously observed in the EW-derived cEGF-treated group, to day 17 (Table 3), because the cEGF was only partially purified, and thus, EW components such as OV may have induced inflammation. We will improve our purification procedure. DISCUSSION The germline transmission efficiency we observed in this present study was similar to that of our previous report (8) (92.2 vs. 95.2% on average, regardless of the transgene used). With the 23ERE-OVcEGF transgene, individual variation was evident when 2 germline chimeric roosters were compared (74.1% for #9056 vs. 37.3% for #9058), but the average efficiency of transgenic chicken production was also similar to that of our previous report (8) (47.1 vs. 52.2%). However, surprisingly, the GFP-OVcEGF transgene was transmitted more efficiently; the average value was 92.3% with a range of 83.3–100.0%. GFP-OVcEGF transgenic chicks were screened using a fluorescent excitation lamp fitted with appropriate filters; this eliminates any possible technical error such as nonspecificity of 8

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genomic PCR amplification. It remains possible that GFPOVcEGF-transfected PGC sublines may have several transgene copies or that heterologous PGC populations have transgenes at various sites. The conditions and factors affecting germline transmission require further investigation. The hen egg is the best model system for an animal bioreactor because genetically selected commercial hens lay eggs almost every day, with ;6.5 g of protein in each egg (22). Particularly, OV (a major EW protein) constitutes ;54% (;2 g) of total protein (21). Thus, the tubular gland cells of the magnum compartment of the oviduct constantly produce massive quantities of EW proteins. Zhu et al. (23) developed somatic chimeras using chicken embryonic stem cells expressing a mouse mAb and assayed the in vitro bioactivity of mAb purified from EW. Although transgenic offspring were not successfully generated via germline transmission, the cited report nonetheless demonstrates the industrial applicability of the transgenic hen as an animal bioreactor. Because transgenes become stably integrated, virusmediated transgenesis methods have been used to produce transgenic chicken bioreactors; several reports on production of bioactive proteins in the EW of transgenic birds generated via germline transmission have appeared (22, 24–26). Harvey et al. (22) produced transgenic chickens expressing b-lactamase using a replication-deficient retroviral ALV. The cited authors maintained transgenic lines for 4 generations; no transgene silencing was evident. G2 transgenic hens expressed EW b-lactamase at concentrations ranging from 0.47 to 1.34 mg/ml and G3 hens at


PARK ET AL.

Figure 5. hEGF expression levels in transgenic hen EW and bioactivity assay of EW-derived EGF. The expression levels of hEGF in transgenic hen EW of (A) heterozygous G1 and (B) homozygous G2 lines are shown. In vivo assay of the effect of EW-derived hEGF on wounded skin of the miniature pig is shown. C) In vitro proliferation assay using EW-derived hEGF is shown. The bioactivity of EW-derived hEGF on HeLa cells was compared to that of EW alone and commercial hEGF. The hEGF concentration was adjusted to 1 ng/ml, and all tests featured 3 replicates per treatment. Both EW-derived and commercial hEGF significantly enhanced HeLa cell proliferation, compared to EW alone (*P , 0.05). D) H&E staining of paraffin-embedded sections was performed on days 3, 7, and 14 postwounding, and MT staining was performed on day 14.

concentrations ranging from 0.52 to 1.65 mg/ml (22). The average expression level in homozygous G3 hens was 47% higher than that in heterozygous hens. Kamihira et al. (24) generated scFV-Fc (single-chain Fv-Fc fusion protein)expressing transgenic chickens that deposited human Ig in EW at levels ranging from 0.1 to 1.5 mg/ml. Lillico et al.

(25) created 2 transgenic lines expressing hIFNb1a and a humanized ScFv-Fc miniantibody (miR24). The miR24 antibody was deposited in transgenic EW at levels ranging from 3.5 to 426 mg/ml, and recombinant hIFNb1a protein accumulated to a mean level of 38 mg/ml (25). In our previous report, recombinant human IL-1 receptor

TABLE 3. Summary of microscopic findings in wounded skin treated with EW-derived EGF Microscopic findings Inflammation Group

EW-EGF

No EGF

Fibroplasia

Neovascularization

Re-epithelialization

Collagen synthesis

Animal number

d3

d7

d 14

d3

d7

d 14

d3

d7

d 14

d3

d7

d 14

d3

d7

d 14

1 2 3 4 Mean 1 2 3 4 Mean

3 3 3 3 3.0 3 3 3 3 3.0

3 2 2 2 2.3 1 3 2 3 2.3

1 2 1 1 1.3 2 1 1 1 1.3

0 0 0 0 0 0 0 0 0 0

3 3 2 3 2.8 3 1 3 2 2.3

2 2 2 2 2.0 2 2 1 2 1.8

0 0 0 0 0 0 0 0 0 0

3 3 3 2 2.8 1 3 2 3 2.3

1 2 2 1 1.5 2 1 1 2 1.5

0 0 0 0 0 0 0 0 0 0

1 1 1 2 1.3 3 1 1 1 1.5

3 3 3 3 3.0 3 2 3 3 2.8

0 0 0 0 0 0 0 0 0 0

1 1 1 2 1.3 1 1 1 1 1.0

2 2 2 2 2.0 2 2 2 2 2.0

0, no remarkable lesion; 1, mild; 2, moderate; 3, severe (definition of each criterion is described in Materials and Methods).


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antagonist (rhIL1RN) was successfully deposited in transgenic quail EW, and an in vitro assay showed that EWderived rhIL1RN was biofunctional (26). Although the viral transduction is the most convincing strategy to generate the transgenic poultry, the safety issue of virus-mediated gene transfer would hinder industrial applications. Recently, nonviral transposons with high transgene insertion efficacy have been utilized to create the transgenic chickens (7, 8). In the present study, to verify the transposonmediated gene transfer system to produce bioactive material, we generated transgenic hens specifically expressing and depositing hEGF in their eggs and, additionally, assayed the bioactivity thereof using an in vivo model featuring miniature pigs. In the present study, a 3.0 kb DNA segment bearing the chicken OV promoter, including the 59-UTR, contained several regulatory elements: a steroid-dependent regulatory element (SDRE), negative regulatory element (NRE), and chicken OV gene upstream promoter (COUP). A transcriptional initiation site, a TATA box, was also present. In addition, we ligated 23ERE to the chicken OV promoter to enhance the specificities for, and the expression levels of transgenes in, the oviduct. The SDRE, COUP, and EREs upregulated the expression of chicken cEGF, whereas the negative element, NRE, facilitated oviduct-specific expression. We used a 1.6 kb segment of the OV 39-UTR containing poly-A sequences, rather than a conventional poly-A tail. The EGF transgene was successfully driven and regulated by the minisynthetic promoter, and EGF was secreted with the aid of the chicken lysozyme signal peptide. EGF expressed in many human tissues stimulates cell growth, proliferation, and differentiation, via signal transduction through the EGF receptor. hEGF is a 6 kDa low molecular weight polypeptide of 53 amino acids featuring 3 intramolecular disulfide bonds (27, 28). Because EGF acts as a potent mitogenic factor, and plays important roles in the growth and proliferation of many cell types, inhibitors of the signaling pathways of EGF or the EGF receptor have been developed as treatments for certain types of cancer (28). Recently, however, EGF has also found industrial applications, for example, as a cosmeceutical. In the present study, the EGF expression levels in the EWs of transgenic hens were lower than those (of other materials) described in previous reports. This is because EGF is relatively small, compared to enzymes and antibodies. In the next step of our work, we will increase transgene expression by modifying promoter regulation and via insertion of multiple copies of the cEGF gene. However, hEGF deposited in EW significantly enhanced HeLa cell proliferation in vitro, to an extent similar to that of commercial hEGF, and was also bioactive, functioning to enhance reepithelialization via fibroplasia and neovascularization of wounded skin in the in vivo miniature pig model. Additionally, in further studies, the purification efficiency of hEGF should be improved to eliminate the contamination of the EW proteins caused by the negative inflammatory effects. Recently, we generated the OV knockout chicken (21), and the genetically modified chicken line could be utilized to enhance the purification process efficiency and yield of the target protein from chicken EW. In conclusion, our results suggest that the transgenic chicken bioreactor system could be of great use to the agricultural and pharmaceutical industries 10

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and could be employed for efficient production of various biomaterials. This work was supported by the Bio-industry Technology Development Program (IPET-312060-5-2-SB020), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported by the Technological Innovation R&D Program (SA112684) funded by the Small and Medium Business Administration (Korea).

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