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. 2021 Jun 29;100(10):101365. doi: 10.1016/j.psj.2021.101365

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells

Hyeon Yang 1, Bo Ram Lee 1, Hwi-Cheul Lee 1, Hoonsung Choi 1, Sun Keun Jung 1, Ji-Youn Kim 1, Jingu No 1, Sureshkumar Shanmugam 1, Yong Jin Jo 1, Keon Bong Oh 1, Kyung Woon Kim 1, Sung June Byun 1,1
PMCID: PMC8358702  PMID: 34375836

Abstract

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

Key words: chicken, ovalbumin promoter, recombinant promoter, reporter analysis

INTRODUCTION

Recombinant proteins are widely produced in diverse platforms, such as microorganisms, animal cell lines, plants, and animals (Zhu, 2012). Avian species have several advantages over other platforms as bioreactors to produce recombinant proteins because of the benefits of glycosylation and posttranslational modification (Lillico et al., 2005; Kojima et al., 2014). The low cost, efficient scale-up, and relatively simple purification of the proteins are feasible in avian bioreactor systems (Ivarie, 2006; Lillico et al., 2007). Therefore, many transgenic chickens that produce recombinant proteins were generated. These chickens recently exhibited meaningful performances and received approval from the Food and Drug Administration (FDA) (Sheridan, 2016).

Unique approaches for producing transgenic avian species were used because of their species-specific reproductive mechanism, and virus injection into the subgerminal cavity of Eyal-giladi and Kochav Stage-X (EGK-X) chicken embryos is a well-defined method (McGrew et al., 2004). This system uses an ovalbumin gene promoter to induce transgene expression in transgenic chickens due to its oviduct-specific regulation and productivity. Four DNase I-hypersensitive sites (DHSs) are located between the ovalbumin locus and its upstream neighboring gene (Kaye et al., 1986), and the estrogen-responsive enhancer element (ERE) DHS III is responsible for estrogen inducibility and regulation of oviduct-specific expression (Kato et al., 1992; Kodama et al., 2012). The estrogen hormone and receptor complex activates transcription of the target gene via binding to the ERE in the promoter of the gene (Giguere, 1999). Steroid-dependent regulatory elements (SDREs) and negative-dependent regulatory elements (NREs) in the 5`-flanking region of the ovalbumin transcription start site functionally collaborate for oviduct-specific gene expression and regulation (Dougherty and Sanders, 2005).

Several types of ovalbumin promoters were used to construct transgene expression cassettes in viral vectors, and a higher level of recombinant protein expression, such as human functional cytokines and lysozyme, was observed when the ovalbumin ERE was placed upstream of the ovalbumin promoter (Lillico et al., 2007; Cao et al., 2015; Herron et al., 2018). Although a reconstructed ovalbumin promoter that links an ovalbumin promoter and the ERE is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. Lentivirus titers are significantly decreased for large transfer vectors (Kumar et al., 2001; Yacoub et al., 2007). Therefore, the production of chimeric chickens with germline transmission of a transgene may have low efficiency following injection of a low-titer virus into EGK-X embryos.

The present study newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. The recombinant promoters were designed to be more compact than the reconstructed ovalbumin promoter, and these constructs were evaluated in several human and chicken somatic cells and cultured primary chicken oviduct cells.

MATERIALS AND METHODS

Experimental Animals and Animal Care

The experimental protocol used in this study was performed under an approved animal-use document and according to the animal care and use guidelines of the committee of the National Institute of Animal Science, Republic of Korea (approval no: 2017-219).

Cell Preparation and Culture

Human cervix epithelial (HeLa and CCL-2; ATCC), human ovarian epithelial (MES-SA and CRL-1976; ATCC), and chicken liver epithelial (Leghorn male hepatoma [LMH]/2A and CRL-2118; ATCC) cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured, and stocked according to the manufacturer's instructions. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% antibiotic/antimycotic (Gibco, Carlsbad, CA). Chicken embryonic fibroblast cells (cEFs) were trypsinized and collected via centrifugation (Schmid et al., 1983), and the DF-1 chicken embryonic fibroblast cell line (CRL-12203; ATCC) was cultured in DMEM supplemented with 10% FBS, 1% antibiotic/antimycotic, and 2% chicken serum (Gibco, Carlsbad, CA). Chicken oviduct epithelial cells (cOECs) were isolated and cultured according to a previous study (Kasperczyk et al., 2012; Yang et al., 2021). Briefly, the magnum from a 35-wk egg-laying hen (White Leghorn) oviduct was surgically separated, and the tissue was horizontally torn off to expose the inner surface. The inner surface was scraped, and the scraped tissue fragments were minced, digested with 10 mL (1 mg/mL) of collagenase P (Roche, Basel, Switzerland) supplemented with 10 mM HEPES (Sigma-Aldrich, Saint Louis, MO), and placed in an incubator with 5% CO2 at 37°C for 30 min with vortexing every 5 min. The digested tissue masses were centrifuged and cultured on collagen-treated dishes (Corning, New York, NY) with keratinocyte complete medium (K-SFM; Gibco, Carlsbad, CA) supplemented with 5% chicken serum and 1% antibiotic/antimycotic.

Vector Construction

As a control, a reconstructed ovalbumin promoter (Mut-4.4-kb-pOV) linking the 2.8-kb ovalbumin promoter and the 1.6-kb ovalbumin ERE was synthesized (Herron et al., 2018). Two restriction enzyme sites (Nhe I and Xho I) were inserted at the end of the 5′- and 3′-flanking regions via PCR amplification, and the promoter was cloned into the pGL4.11/luc2p firefly DNA vector (Promega, Madison, WA). Recombinant promoters linking putative enhancer elements of chicken oviduct-expressing genes, namely ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), to the 5′-flanking region of the 2.8-kb ovalbumin promoter were synthesized using 2 restriction enzyme sites (Nhe I and Xho I). Recombinant promoters linking basal promoters of the genes, namely pTF, lysozyme (pLYZ), and ovomucoid (pOVM), to the 3`-flanking region of the 1.6-kb ovalbumin ERE were synthesized using 2 restriction enzyme sites (Xho I and EcoR V). These 6 recombinant promoters were cloned into the pGL4.11 reporter vectors.

Transfection

Each pGL4.11 reporter vector cloned with Mut_4.4-kb_pOV and the 6 recombinant promoters were used together with the pGL4.74/hRluc Renilla DNA vector (Promega, Madison, WA). Renilla is driven by the herpes simplex virus thymidine kinase promoter. pGL4.11 and pGL4.74 were cotransfected into HeLa cells, MES-SA cells, cEFs, LMH/2A cells, and cOECs using a Lipofectamine 3,000 kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, cells were seeded into a 12-well culture plate, and transfection was performed at 70% cell density, considering the molecular ratio of each vector. To perform transfection, 50 µL of Opti-MEM (Gibco, Carlsbad, CA) containing 1 µg of pGL4.11, 0.01 µg of pGL4.74, and 2 µL of P3,000 per well and another 50 µL of Opti-MEM containing 1.5 µL of Lipofectamine reagent per well were mixed for 20 min at room temperature. A volume of 100 µL of the mixture per well was added to 12-well culture plates, and the culture plate was placed in an incubator with 5% CO2 at 37°C for 24 h.

Luciferase Assay

To analyze the promoter activity, a dual luciferase assay was performed. The cell culture medium was removed, and 250 µL of passive lysis buffer was added to each well of the 12-well culture plate. The plate was stirred for 20 to 30 min to lyse the cells. The lysed cells were transferred into 1.5-mL E-tubes, which were centrifuged at 12,000 rpm for 10 min, and 20 µL of each lysate was transferred to a 96-well white microplate (Nunc, Rochester, NY). To analyze the estrogen response reaction in LMH/2A cells, the cells were additionally cultured for 24 h with medium containing 0-1,000 nM 17β-estradiol (Sigma-Aldrich, Saint Louis, MO) at 24 h post transfection. These cells were prepared as described above. Each luciferase activity was measured using a Centro LB 960 luminometer (Berthold, Bad Wildbad, Germany). The normalized luciferase value was calculated as the firefly luminescence/Renilla luminescence ratio. The relative luciferase ratio for promoter activity was recombinant promoter luciferase ratio/Mut-4.4-kb-pOV luciferase ratio. The relative luciferase ratio for the estrogen response was estrogen treatment luciferase ratio/non-estrogen treatment luciferase ratio.

Immunofluorescence

cOECs at passage 1 were seeded into a 4-well culture plate, fixed with 4% paraformaldehyde (Molecular Probes, Eugene, OR), and permeabilized with 0.5% Triton-X (Gibco, Carlsbad, CA) for 15 min at room temperature. Cells were blocked with 5% bovine serum albumin (BSA; Molecular Probes, Eugene, OR) for 1 h at room temperature and incubated with a primary rabbit polyclonal anti-ovalbumin antibody (1:250; 1 mg/mL; Abcam, Cambridge, UK) overnight at 4°C. Cells were incubated with a secondary mouse anti-rabbit FITC conjugated antibody (1:500; 2 mg/mL; Abcam) for 1 h and washed with PBS (Gibco, Carlsbad, CA). Cells were counterstained for DNA using Hoechst 33258 (1:4,000; 10 mg/mL; Life Technologies, Carlsbad, CA). Immunofluorescence was analyzed under a fluorescence microscope.

Western Blot Analysis

Total protein was extracted from the chicken oviduct as a positive control and from chicken leg tissue and embryonic fibroblast cells (DF-1) as negative controls. Total protein was also extracted from the cOECs. RIPA lysis buffer (Thermo Scientific, Waltham, MA) containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA) was used for protein extraction. The protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA), and 15 μg of protein extract was electrophoresed using an SDS-PAGE 4 to 12% gel system (Invitrogen, Carlsbad, CA). Separated proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Invitrogen, Carlsbad, CA). The membrane was blocked with 5% skim milk in PBS (Thermo Scientific, Waltham, MA) for 1 h and incubated with a rabbit polyclonal anti-ovalbumin antibody (1:1,000; 1 mg/mL; Abcam, Cambridge, UK) and rabbit monoclonal anti-vinculin antibody (1:1,000; 0.054 mg/mL; Abcam, Cambridge, UK) overnight at 4°C. The membrane was washed and incubated with a mouse anti-rabbit HRP-conjugated antibody (1:2,000; 0.4 mg/mL; Santa Cruz, Dallas, TX) for 30 min at room temperature. Amersham ECL prime (GE Healthcare, Buckinghamshire, UK) substrate was used to visualize the target bands, and the bands were analyzed using EZ-Capture II (Atto, Tokyo, Japan).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism statistical software (GraphPad Prism 5.03 software, San Diego, CA). One-way ANOVA was used to compare the relative luciferase ratio, and a P-value less than 0.05 was considered statistically significant. The reporter analysis data are expressed as the mean ± standard error of the mean from three independent experiments.

RESULTS

Construction of the Recombinant Chicken Promoters

To develop chicken recombinant promoters to efficiently drive transgene expression, a reconstructed ovalbumin promoter (Mut_4.4-kb_pOV) was used as a control in reporter analysis, and 6 recombinant promoters (TF_pOV, OVOA_pOV, OVALX_pOV, ERE_pTF, ERE_pLYZ, and ERE_pOVM) were synthesized and cloned into the pGL4.11 reporter vector. The Mut_4.4-kb_pOV structure is shown in Figure 1, and the structures of the 6 recombinant promoters are shown in Figure 2. Notably, 958 bp of TF, 977 bp of OVOA, and 1,468 bp of OVALX were linked upstream of the 2.8-kb ovalbumin promoter (Figure 2A), then 1,126 bp of pTF, 857 bp of pLYZ, and 1,350 bp of pOVM were linked downstream of the 1.6-kb ovalbumin ERE (Figure 2B). These recombinant sequences, except pLYZ, had several putative estrogen receptor (ER) alpha binding sites as follows: 5 (1.6-kb ovalbumin ERE), 3 (2.8-kb ovalbumin promoter), 2 (TF), 2 (OVOA), 3 (OVALX), 1 (pTF), and 1 (pOVM) in the direction of 5` to 3` (Table 1). As a result, the lengths of the recombinant promoters were 3,742 (TF_pOV), 3,761 (OVOA_pOV), 4,252 (OVALX_pOV), 2,778 (ERE_pTF), 2,509 (ERE_pLYZ), and 3,002 bp (ERE_pOVM). Therefore, the recombinant promoters were more compact than Mut_4.4-kb_pOV (4,436 bp) and may have oviduct-specific regulation and expression via the ovalbumin regulatory regions, 2.8-kb ovalbumin promoter or 1.6-kb ovalbumin ERE.

Figure 1.

Figure 1

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Structure of the reconstructed chicken ovalbumin promoters. Mut_4.4-kb_pOV with a 1-kb deletion between the 1.6-kb ovalbumin ERE and the 2.8-kb ovalbumin promoter in the ovalbumin locus was constructed via gene synthesis for use as a control in the luciferase assay. Nucleotide sequences are denoted based on the translation start site (ATG) as a +1. Abbreviations: DHS III, DNase hypersensitive site III; NRE, negative-dependent regulatory element; SDRE, steroid-dependent regulatory element.

Figure 2.

Figure 2

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Structures of the six recombinant promoters constructed by linking regulatory regions of ovalbumin and other oviduct-specific genes. (A) Chicken oviduct expression genes (958 bp of TF, 977 bp of OVOA, and 1,468 bp of OVALX) were linked upstream of the 2.8-kb ovalbumin promoter. (B) In addition, 1,126 of pTF, 857 bp of pLYZ, and 1,350 bp of pOVM were linked downstream of the 1.6-kb ovalbumin ERE. Nucleotide sequences are denoted based on the translation start site (ATG) as a +1. Abbreviations: LYZ, lysozyme; OVOA, ovomucin alpha subunit; OVALX, ovalbumin-related protein X; OVM, ovomucoid; TF, ovotransferrin.

Table 1.

Prediction of putative estrogen receptor alpha-binding sites.

Name Sequences (5` to 3`) Start position End position Dissimilarity
1.6-kb ERE TTGACCTGATA -5,395 -5,385 0.000000
GTGACCTGAGC -5,268 -5,258 0.722887
TTGACCTGTGA -5,068 -5,058 0.000000
CTGACCTTTTC -4,916 -4,906 2.007569
CTGACCTCAGT -3,928 -3,918 4.617205
2.8-kb pOV ACAGAGGTCAG -2,568 -2,558 4.617205
TAATAGGTCAC -2,138 -2,128 3.734241
TCAAAGGTCAA -1,735 -1,725 2.007569
TF TTGACCTATTG -4,505 -4,495 4.617205
GTGACCTGACC -4,494 -4,484 2.328738
OVOA GTGACCTGGCA -4,711 -4,701 3.934590
CAATAGGTCAA -4,156 -4,146 4.617205
OVALX CTGACCTCTTT -4,389 -4,379 4.617205
CTGACCTGTAG -3,763 -3,753 3.211703
ATGACCTATGA -3,107 -3,097 4.457128
pTF CGTTAGGTCAT -150 -140 7.668831
pLYZ - - - -
pOVM CTGGAGGTCAG -186 -176 7.828908
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Nucleotide sequences are denoted based on translation start site (ATG) as a +1.

Activity and Estrogen Response of the Recombinant Chicken Promoters in Somatic Cells

To validate the characteristics of the recombinant promoters, such as promoter activity, cell-specificity, and estrogen responsivity, each reporter vector cloned with Mut_4.4-kb_pOV and the 6 recombinant promoters were transfected into HeLa cells, MES-SA cells, cEFs and LMH/2A cells. A dual luciferase assay was performed to evaluate the relative promoter activity in these cells (Figure 3). Although the promoter activities of TF_pOV and OVOA_pOV showed no large differences from each other in HeLa cells (Figure 3A), OVOA_pOV had 2.0-fold (P < 0.01) higher activity in MES-SA cells compared to the control (Figure 3B). There were no significant differences between all groups in chicken embryonic fibroblast cells (Figure 3C). TF_pOV and OVOA_pOV showed 2.1- (P < 0.05) and 1.7-fold (P > 0.05), respectively, higher relative luciferase expression in LMH/2A cells compared to the control (Figure 3D). However, ERE_pLYZ and ERE_pOVM showed no significant differences compared with the control, but ERE_pTF had significantly higher luciferase activity in all cells, especially in chicken embryonic fibroblast cells (Figure 3E–3H). The seven reporter vectors mentioned above were transfected into LMH/2A cells followed by treatments with 0 to 1,000 nM estrogen 24 h post-transfection. A dual luciferase assay was performed to investigate the estrogen response at 24 h posttreatment (Figure 4). The luciferase activity was significantly increased 2.5-fold (P < 0.01) after treatment with 1,000 nM estrogen in the control (Figure 4A). TF_pOV and OVOA_pOV showed 56.4- (P < 0.01) and 15.9-fold (P < 0.01) increases, respectively, with 1,000 nM estrogen treatments (Figure 4B, 4C), but there were no increases following estrogen treatments in OVALX_pOV or ERE_pTF (Figure 4D, 4E), and only 2.0- (P < 0.05) and 1.4-fold (P < 0.001) increases were confirmed in ERE_pLYZ and ERE_pOVM, respectively (Figure 4F, 4G). Taken together, TF_pOV, OVOA_pOV, ERE_pLYZ, and ERE_pOVM were considered to have the potential to efficiently drive transgene expression, and these constructs were selected for additional evaluation of promoter activity using the dual luciferase assay in cultured primary cOECs.

Figure 3.

Figure 3

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Relative luciferase ratio of recombinant promoters in human and chicken somatic cells. Each reporter vector cloned with a control and the six recombinant promoters was transiently transfected into HeLa cells (A, E), MES-SA cells (B, F), cEFs (C, G), and LMH/2A cells (D, H). Luciferase assays were performed 24 h post-transfection. Data represent relative values obtained with Mut_4.4-kb_pOV. The normalized luciferase value was calculated as firefly luminescence/Renilla luminescence ratio, and the relative luciferase ratio was calculated as recombinant promoter luciferase ratio/Mut_4.4-kb_pOV luciferase ratio. Error bars indicate the mean ± SEM (n = 3). * P < 0.05, ⁎⁎P < 0.01, and ⁎⁎⁎P < 0.001 compared to Mut_4.4-kb_pOV.

Figure 4.

Figure 4

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Estrogen responsivity of recombinant promoters following estrogen treatments in LMH/2A cells. Each reporter vector cloned with a control and the six recombinant promoters was transiently transfected into LMH/2A cells followed by treatment with 0, 1, 10, 100, and 1,000 nM estrogen 24 h post-transfection. Luciferase assays were performed 24 h after estrogen treatments. Data represent the relative values obtained with 0 nM (non-E2) estrogen treatment in Mut_4.4-kb_pOV (A), TF_pOV (B), OVOA_pOV (C), OVALX_pOV (D), ERE_pTF (E), ERE_pLYZ (F), and ERE_pOVM (G). The normalized luciferase value was calculated as firefly luminescence/Renilla luminescence ratio, and the relative luciferase ratio was calculated as estrogen treatment luciferase ratio/non-estrogen treatment luciferase ratio. Error bars indicate the mean ± SEM (n = 3). * P < 0.05, ⁎⁎P < 0.01, and ⁎⁎⁎P < 0.001 compared to 0 nM estrogen treatment.

Activity of the Recombinant Chicken Promoters in Chicken Oviduct Cells

To additionally evaluate the relative promoter activity of the recombinant promoters, TF_pOV, OVOA_pOV, ERE_pLYZ, and ERE_pOVM, we isolated and cultured primary chicken oviduct cells from a 35-wk egg-laying hens, and the cells were confirmed to express ovalbumin protein using immunofluorescence (Figure 5A). Western blot analysis indicated that chicken ovalbumin protein was clearly detected in the cOECs and oviduct tissue but not in chicken leg tissue or chicken embryonic fibroblast cells (Figure 5B). Each reporter vector was cloned with Mut_4.4-kb_pOV and four recombinant promoters (TF_pOV, OVOA_pOV, ERE_pLYZ, and ERE_pOVM) were transfected into the cOECs. A dual luciferase assay was performed to compare relative promoter activities. Comparative luciferase analysis showed that TF_pOV, OVOA_pOV, ERE_pLYZ, and ERE_pOVM had 2.1- (P < 0.001), 2.3- (P < 0.05), 19.5- (P < 0.05), and 4.0-fold (P < 0.001) higher activity than the control in the cOECs, respectively (Figure 5C–5F). Consequently, recombinant promoters linking ovalbumin and TF, OVOA, pOVM, andpLYZ regulatory regions showed higher or similar levels of promoter activity in somatic cells and higher levels of estrogen response in LMH/2A cells. These constructs also showed significantly higher promoter activity in chicken oviduct cells. These results confirmed that reconstitution with regulatory regions of ovalbumin and other oviduct-specific genes contributed to an efficient driving of transgene expression in chicken oviduct cells.

Figure 5.

Figure 5

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Characterization of chicken oviduct cells and relative luciferase ratio of the recombinant promoters in cells. (A) Primary chicken oviduct epithelial cells were cultured. Cell morphology at passage 1 (a, b) and immunofluorescence images for nuclei (c) and ovalbumin (d) at passage 2 are presented. Scale bars: 100 µm. (B) Western blot analysis of oviduct epithelial cells. Chicken oviduct magnum (pc, positive control), chicken leg muscle tissue and DF-1 (nc, negative control) are shown. Ovalbumin (OVA) and vinculin (VCL) proteins were used as oviduct-specific markers and loading controls. (C–F) Relative luciferase ratios of the TF_pOV (C), OVOA_pOV (D), ERE_pLYZ (E), and ERE_pOVM (F) recombinant promoters in oviduct cells are shown. Luciferase assays were performed at 24 h post transfection. Data represent the relative values obtained with Mut_4.4-kb_pOV. The normalized luciferase value was calculated as firefly luminescence/Renilla luminescence ratio, and the relative luciferase ratio was calculated as recombinant promoter luciferase ratio/Mut_4.4-kb_pOV luciferase ratio. Error bars indicate the mean ± SEM (n = 3). * P < 0.05, ⁎⁎P < 0.01, and ⁎⁎⁎P < 0.001 compared to Mut_4.4-kb_pOV.

DISCUSSION

The present study newly developed recombinant chicken promoters, and these recombinant promoters were evaluated in human and chicken somatic cells and cultured primary chicken oviduct cells with a dual luciferase assay. Virus injection into EGK-X chickens and quail embryos and the transfer of primordial germ cells (PGCs) into blood vessels are widely used in avian transgenesis (van de Lavoir et al., 2006; Lee et al., 2019). Although PGC-mediated transgenesis requires establishment via isolation, the in vitro culture, characterization, modification, selection and transfer of PGCs, virus injection into blastoderm cells is a relatively simple process.

The recombinant viral particles used for injection to generate transgenic chickens that produce recombinant proteins are constructed using packaging and transfer plasmids, including a transgene and promoter. The packaged viral particles are injected into EGK-X embryos. Several types of ubiquitous promoters, such as cytomegalovirus, β-actin, rous sarcoma virus, and human phosphoglycerate promoter, were reported in viral vector constructs (Woodfint et al., 2018). Although the constitutive promoter CMV induced a high concentration of human erythropoietin (hEPO) in chicken blood, pathophysiological abnormalities such as reddish appearance, vasodilation, and death at an early stage, occur (Koo et al., 2017). However, these unfavorable phenotypes were improved by driving hEPO under the control of the chicken ovalbumin promoter (Kwon et al., 2018). Accordingly, oviduct-specific promoters for chicken bioreactors may be a solution to uncontrolled and unexpected side effects.

SDREs, NREs, and EREs are located in the 5`-flanking regions of the ovalbumin transcription start site. The chicken ovalbumin promoter has a translation start site (ATG) at the beginning of exon 2, and the 2.8-kb ovalbumin promoter consists of SDRE, NRE, exon 1, intron 1, and the beginning of exon 2. The use of this 2.8-kb ovalbumin promoter with a 0.7- to 1.6-kb ERE induced higher expression levels of transgenes (Lillico et al., 2007; Cao et al., 2015; Herron et al., 2018) compared to our previous study, in which green fluorescence protein or extracellular human superoxide dismutase was driven by ovalbumin promoters without intron 1 (Byun et al., 2011; Byun et al., 2013). The 6 recombinant promoters in the present study were constructed based on these regulatory elements and had a more compact size (184- to 1,928-bp) than Mut_4.4-kb_pOV, which may enable a wider selection of a target gene and efficient processing for lentivirus packaging.

The relative luciferase activity ratio under the control of the 6 recombinant promoters was analyzed in HeLa cells, MES-SA cells, cEFs, LMH/2A cells, and cOECs compared to Mut_4.4-kb_pOV. HeLa and MES-SA cells are somatic epithelial cells from the human cervix and ovarian cancer, respectively, and these cells were used to perform a comparative analysis of promoter activity between recombinant promoters and the control in somatic epithelial cells. Chicken embryonic fibroblast cells (cEFs), were used to analyze cell-specificity. LMH/2A was used to perform the comparative analysis of promoter activity in somatic epithelial cells and investigate estrogen responsivity under the control of all promoters following estrogen treatments. The recombinant promoters shared one promoter organization, such as the 2.8-kb ovalbumin promoter or 1.6-kb ovalbumin ERE. Therefore, their ability to induce luciferase activity was expected to be similar in somatic cells. However, there were significant differences according to the promoter type and somatic cells type.

TF_pOV and OVOA_pOV had significantly higher expression levels in LMH/2A cells, and moderate expression levels in HeLa cells in the present study. OVOA_pOV showed significantly higher expression levels in MES-SA cells. The results showed that these regulatory element of 958 bp of TF and 1,002 bp of OVOA effectively induced luciferase gene expression in somatic epithelial cells compared to DHS III in the control. However, OVALX_pOV was a less effective organization than TF_pOV and OVOA_pOV, due to its weak performance. ERE_pLYZ and ERE_pOVM also showed no large differences, but ERE_pTF showed significantly higher activity in the cells, especially 89.8-fold in cEFs compared to the control. Because ERE_pTF was thought to have lost tissue-specific regulation, it was excluded from subsequent validation in oviduct cells. As a result, significant differences in luciferase expression levels may be caused by interactions between cell-specific transcription factors and nucleotide sequences in regulatory regions.

LMH/2A cells are a chicken liver epithelial cell line that originated from the parental LMH cell line established by the integration of the chicken ER alpha expression vector (Binder et al., 1990), and ovalbumin promoters function in chicken embryonic hepatocytes (Dierich et al., 1987). Therefore, the LMH/2A cell line was additionally used to validate the estrogen response for all recombinant promoters. The recombinant promoters in this study were designed to have at least one ER alpha binding site, as predicted with PROMO 3.0 (Messeguer et al., 2002). The reconstructed ovalbumin promoter (Mut_4.4-kb_pOV) clearly showed an increase after estrogen treatment. TF_pOV, OVOA_pOV and OVALX_pOV, which consist of a 2.8-kb ovalbumin promoter, showed different estrogen responsivities, which indicates that regulatory regions, such as ERE, TF, OVOA, or OVALX, may determine the estrogen effect. However, ERE_pTF did not show a sufficient increase with estrogen treatment, despite small increases in ERE_pLYZ and ERE_pOVM. To the best of our knowledge, this discordance of results may be affected by a misguided prediction of ER alpha binding sites in regulatory regions or accidental inhibitory effects of the reconstitution of nucleotide sequences from different genes.

Chicken ovalbumin genes are specifically expressed and regulated in chicken oviduct tissue. Therefore, the newly developed ovalbumin promoters should be validated in chicken oviduct cells. The isolation, in vitro culture, and characterization of cOECs were reported recently (Jung et al., 2011; Kasperczyk et al., 2012; Stadnicka et al., 2018). We also previously established the isolation and in vitro culture of these cells, and the cells were maintained at least until passage 5 (Yang et al., 2021). The TF_pOV, OVOA_pOV, ERE_pLYZ, and ERE_pOVM recombinant promoters showed 2.1- to 19.5-fold higher luciferase activity than the control in the present study, but increases in luciferase activity following estrogen treatments were not confirmed (data not shown). The long-term in vitro culture of the cells remains challenging. Therefore, stably established chicken oviduct cell lines are required to deeply investigate oviduct cell biology and its applications.

Synthetic recombinant promoters linking regulatory regions of ovalbumin and other oviduct-specific genes, such as TF, OVOA, LYZ, and ovomucoid, efficiently drove higher transgene expression in chicken oviduct cells. In conclusion, TF_pOV and ERE_pLYZ were expected to be used to generate lentivirus particles, and application in further avian transgenic studies. These findings provide an understanding of the potential to reconstitute chicken oviduct-expressing genes and produce recombinant proteins, such as pharmaceuticals, in transgenic chickens.

ACKNOWLEDGMENTS

This work was supported by the National Institute of Animal Science (Grant No. PJ01260301), Rural Development Administration (RDA), Republic of Korea.

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

REFERENCES

  1. Binder R., MacDonald C.C., Burch J.B., Lazier C.B., Williams D.L. Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line. Mol. Endocrinol. 1990;4:201–208. doi: 10.1210/mend-4-2-201. [DOI] [PubMed] [Google Scholar]
  2. Byun S.J., Kim S.W., Kim K.-W., Kim J.S., Hwang I.-S., Chung H.K., Kan I.S., Jeon I.-S., Chang W.-K., Park S.-B., Yoo J.G. Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens. Biosci. Biotechnol. Biochem. 2011;75:646–649. doi: 10.1271/bbb.100721. [DOI] [PubMed] [Google Scholar]
  3. Byun S.J., Ji M.-R., Jang Y.-J., Hwang A.-I., Chung H.K., Kim J.S., Kim K.-W., Chung H.-J., Yang B.-C., Jeon I., Park J.-K., Yoo J.G., Kim T.-Y. Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken. BMB Rep. 2013;46:404–409. doi: 10.5483/BMBRep.2013.46.8.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao D., Wu H., Li Q., Sun Y., Liu T., Fei J., Zhao Y., Wu S., Hu X., Li N. Expression of recombinant human lysozyme in egg whites of transgenic hens. PLoS One. 2015;10 doi: 10.1371/journal.pone.0118626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dierich A., Gaub M.P., LePennec J.P., Astinotti D., Chambon P. Cell-specificity of the chicken ovalbumin and conalbumin promoters. EMBO. J. 1987;6:2305–2312. doi: 10.1002/j.1460-2075.1987.tb02505.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dougherty D.C., Sanders M.M. Estrogen action: revitalization of the chick oviduct model. Trends. Endocrinol. Metab. 2005;16:414–419. doi: 10.1016/j.tem.2005.09.001. [DOI] [PubMed] [Google Scholar]
  7. Giguère V. Orphan nuclear receptors: from gene to function. Endocr. Rev. 1999;20:689–725. doi: 10.1210/edrv.20.5.0378. [DOI] [PubMed] [Google Scholar]
  8. Herron L.R., Pridans C., Turnbull M.L., Smith N., Lillico S., Sherman A., Gilhooley H.J., Wear M., Kurian D., Papadakos G., Digard P., Hume D.A., Gill A.C., Sang H.M. A chicken bioreactor for efficient production of functional cytokines. BMC. Biotechnol. 2018;18:82. doi: 10.1186/s12896-018-0495-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ivarie R. Competitive bioreactor hens on the horizon. Trends. Biotechnol. 2006;24:99–101. doi: 10.1016/j.tibtech.2006.01.004. [DOI] [PubMed] [Google Scholar]
  10. Jung J.G., Park T.S., Kim J.N., Han B.K., Lee S.D., Song G., Han J.Y. Characterization and application of oviductal epithelial cells in vitro in Gallus domesticus. Biol. Reprod. 2011;85:798–807. doi: 10.1095/biolreprod.111.092023. [DOI] [PubMed] [Google Scholar]
  11. Kasperczyk K., Bajek A., Joachimiak R., Walasik K., Marszalek A., Drewa T., Bednarczyk M. In vitro optimization of the Gallus domesticus oviduct epithelial cells culture. Theriogenology. 2012;77:1834–1845. doi: 10.1016/j.theriogenology.2011.12.029. [DOI] [PubMed] [Google Scholar]
  12. Kato S., Tora L., Yamauchi J., Masushige S., Bellard M., Chambon P. A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically. Cell. 1992;68:731–742. doi: 10.1016/0092-8674(92)90148-6. [DOI] [PubMed] [Google Scholar]
  13. Kaye J.S., Pratt-Kaye S., Bellard M., Dretzen G., Bellard F., Chambon P. Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5′-flanking segment of the ovalbumin gene. EMBO. J. 1986;5:277–285. doi: 10.1002/j.1460-2075.1986.tb04210.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kodama D., Nishimiya D., Nishijima K.-I., Okino Y., Inayoshi Y., Kojima Y., Ono K.-I., Motono M., Miyake K., Kawabe Y., Kyogoku K., Yamashita T., Kamihira M., Iijima S. Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system. J. Biosci. Bioeng. 2012;113:146–153. doi: 10.1016/j.jbiosc.2011.10.006. [DOI] [PubMed] [Google Scholar]
  15. Kojima Y., Wakita J., Inayoshi Y., Suzuki R., Yamada Y., Kaneoka H., Nishijima K., Iijima S. Galactosylation of human erythropoietin produced by chimeric chickens expressing galactosyltransferase. J. Biosci. Bioeng. 2014;117:676–679. doi: 10.1016/j.jbiosc.2013.11.010. [DOI] [PubMed] [Google Scholar]
  16. Koo B.C., Kwon M.S., Kim D., Kim S.A., Kim N.H., Kim T. Production of transgenic chicken constitutively expressing human erythropoietin (hEPO): problem with uncontrollable overexpression of hEPO gene. Biotechnol. Bioprocess. Eng. 2017;1:22–29. [Google Scholar]
  17. Kumar M., Keller B., Makalou N., Sutton R.E. Systematic determination of the packaging limit of lentiviral vectors. Hum. Gene Ther. 2001;12:1893–1905. doi: 10.1089/104303401753153947. [DOI] [PubMed] [Google Scholar]
  18. Kwon M.S., Koo B.C., Kim D., Nam Y.H., Cui X.-S., Kim N.-H., Kim T. Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific-manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites. PLoS One. 2018;13 doi: 10.1371/journal.pone.0194721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee. J., Ma J., Lee K. Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail. Proc. Natl. Acad. Sci. U S A. 2019;116:13288–13292. doi: 10.1073/pnas.1903230116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lillico S.G., McGrew M.J., Sherman A., Sang H.M. Transgenic chickens as bioreactors for protein-based drugs. Drug. Discov. Today. 2005;10:191–196. doi: 10.1016/S1359-6446(04)03317-3. [DOI] [PubMed] [Google Scholar]
  21. Lillico S.G., Sherman A., McGrew M.J., Robertson C.D., Smith J., Haslam C., Barnard P., Radcliffe P.A., Mitrophanous K.A., Elliot E.A., Sang H.M. Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc. Natl. Acad. Sci. U S A. 2007;104:1771–1776. doi: 10.1073/pnas.0610401104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McGrew M.J., Sherman A., Ellard F.M., Lillico S.G., Gilhooley H.J., Kingsman A.J., Mitrophanous K.A., Sang H. Efficient production of germline transgenic chickens using lentiviral vectors. EMBO. Rep. 2004;5:728–733. doi: 10.1038/sj.embor.7400171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Messeguer X., Escudero R., Farré D., Núñez O., Martínez J., Albà M.M. PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics. 2002;18:333–334. doi: 10.1093/bioinformatics/18.2.333. [DOI] [PubMed] [Google Scholar]
  24. Schmid C., Steiner T., Froesch E.R. Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken embryonic cells. FEBS. Lett. 1983;161:117–121. doi: 10.1016/0014-5793(83)80742-x. [DOI] [PubMed] [Google Scholar]
  25. Sheridan C. FDA approves ‘farmaceutical’ drug from transgenic chickens. Nat. Biotechnol. 2016;34:117–119. doi: 10.1038/nbt0216-117. [DOI] [PubMed] [Google Scholar]
  26. Stadnicka K., Sławińska A., Dunisławska A., Pain B., Bednarczyk M. Molecular signatures of epithelial oviduct cells of a laying hen (Gallus domesticus) and quail (Coturnix japonica) BMC. Dev. Biol. 2018;18:9. doi: 10.1186/s12861-018-0168-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van de Lavoir M.C., Diamond J.H., Leighton P.A., Mather-Love C., Heyer B.S., Bradshaw R., Kerchner A., Hooi L.T., Gessaro T.M., Swanberg S.E., Delany M.E., Etches R.J. Germline transmission of genetically modified primordial germ cells. Nature. 2006;441:766–769. doi: 10.1038/nature04831. [DOI] [PubMed] [Google Scholar]
  28. Woodfint R.M., Hamlin E., Lee K. Avian bioreactor systems: a review. Mol. Biotechnol. 2018;60:975–983. doi: 10.1007/s12033-018-0128-x. [DOI] [PubMed] [Google Scholar]
  29. Yacoub N.a., Romanowska M., Haritonova N., Foerster J. Optimized production and concentration of lentiviral vectors containing large inserts. J. Gene Med. 2007;9:579–584. doi: 10.1002/jgm.1052. [DOI] [PubMed] [Google Scholar]
  30. Yang H., Lee B.R., Lee H.C., Jung S.K., Kim J.-Y., No J., Shanmugam S., Jo Y.J., Lee H., Hwang S.S., Byun S.J. Isolation and characterization of cultured chicken oviduct epithelial cells and in vitro validation of constructed ovalbumin promoter in these cells. Anim Biosci. 2021;34:1321–1330. doi: 10.5713/ab.20.0627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhu J. Mammalian cell protein expression for biopharmaceutical production. Biotechnol. Adv. 2012;30:1158–1170. doi: 10.1016/j.biotechadv.2011.08.022. [DOI] [PubMed] [Google Scholar]

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