Ovalbumin Gene Research Articles

Long-Lasting, Fine-Tuned Anti-Tumor Activity of Recombinant Listeria monocytogenes Vaccine Is Controlled by Pyroptosis and Necroptosis Regulatory and Effector Molecules.

One of the main objectives of developing new anti-cancer vaccine strategies is to effectively induce CD8+ T cell-mediated anti-tumor immunity. Live recombinant vectors, notably Listeria monocytogenes, have been shown to elicit a robust in vivo CD8+ T-cell response in preclinical settings. Significantly, it has been demonstrated that Listeria induces inflammatory/immunogenic cell death mechanisms such as pyroptosis and necroptosis in immune cells that favorably control immunological responses. Therefore, we postulated that the host's response to Listeria-based vectors and the subsequent induction of CD8+ T cell-mediated immunity would be compromised by the lack of regulatory or effector molecules involved in pyroptosis or necroptosis. To test our hypothesis, we used recombinant L. monocytogenes carrying the ovalbumin gene (LM.OVA) to vaccinate wild-type (WT), caspase-1/11-/-, gsdmd-/-, ripk3-/-, and mlkl-/- C57Bl/6 mice. We performed an in vivo cytotoxicity assay to assess the efficacy of OVA-specific CD8+ T lymphocytes in eliminating target cells in wild-type and genetically deficient backgrounds. Furthermore, we evaluated the specific anti-tumor immune response in mice inoculated with the B16F0 and B16F0.OVA melanoma cell lines. Our findings demonstrated that while caspase-1/11 and GSDMD deficiencies interfere with the rapid control of LM.OVA infection, neither of the KOs seems to contribute to the early activation of OVA-specific CTL responses. In contrast, the individual deficiency of each one of these proteins positively impacts the generation of long-lasting effector CD8+ T cells.

Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools.

Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine.

Electroporation-based Easi-CRISPR yields biallelic insertions of EGFP-HiBiT cassette in immortalized chicken oviduct epithelial cells

Laying hens are an excellent experimental oviduct model for studying reproduction biology. Because chicken oviduct epithelial cells (cOECs) have a crucial role in synthesizing and secreting ovalbumin, laying hens have been regarded an ideal bioreactor for producing pharmaceuticals in egg white through transgene or gene editing of the ovalbumin (OVA) gene. However, related studies in cOECs are largely limited because of the lack of immortalized model cells. In addition, the editing efficiency of conventional CRISPR-HDR knock-in in chicken cells is suboptimal (ranging from 1 to 10%) and remains elevated. Here, primary cOECs were isolated from young laying hens, then infected with a retrovirus vector of human telomerase reverse transcriptase (hTERT), and immortalized cOECs were established. Subsequently, an electroporation-based Easi-CRISPR(Efficient additions with ssDNA inserts-CRISPR) method was adopted to integrate an EGFP-HiBiT cassette into the chicken OVA locus (immediately upstream of the stop codon). The immortalized cOECs reflected the self-renewal capability and phenotype of oviduct epithelial cells. This is because these cells not only maintained stable proliferation and normal karyotype and had no potential for malignant transformation, but also expressed oviduct markers and an epithelial marker and had a morphology similar to that of primary cOECs. EGFP expression was detected in the edited cells through microscopy, flow cytometry, and HiBiT/Western blotting. The EGFP-HiBiT knock-in efficiency reached 27.9% after a single round of electroporation, which was determined through genotyping and DNA sequencing. Two single cell clones contained biallelic insertions of EGFP-HiBiT donor cassettes. In conclusion, our established immortalized cOECs could act as an in vitro cell model for gene editing in chicken, and this electroporation-based Easi-CRISPR strategy will contribute to the generation of avian bioreactors and other gene-edited (GE) birds.

Study of the regulatory elements of the Ovalbumin gene promoter using CRISPR technology in chicken cells

BackgroundHormone-dependent promoters are very efficient in transgene expression. Plasmid-based reporter assays have identified regulatory sequences of the Ovalbumin promoter that are involved in response to estrogen and have shown that the deletion of the steroid-dependent regulatory element (SDRE) and negative regulatory element (NRE) leads to a steroid-independent expression of a reporter. However, the functional roles of these regulatory elements within the native genomic context of the Ovalbumin promoter have not been evaluated.ResultsIn this study, we show that the negative effects of the NRE element on the Ovalbumin gene can be counteracted by CRISPR interference. We also show that the CRISPR-mediated deletion of SDRE and NRE promoter elements in a non-oviduct cell can lead to the significant expression of the Ovalbumin gene. In addition, the targeted knock-in of a transgene reporter in the Ovalbumin coding region and its expression confirms that the truncated promoter of the Ovalbumin gene can be efficiently used for an estrogen-independent expression of a foreign gene.ConclusionsThe methodology applied in this paper allowed the study of promoter regulatory sequences in their native nuclear organization.

Egg white composition, antioxidant capacity, serum and yolk lipids and oxidative damage of the oviduct magnum in laying hens fed diets contaminated with different concentrations of cadmium

Total of 384 healthy 38-week-old hens were randomly divided into four treatments. Layers in four groups were fed a basal diet and a basal diet with Cd at the level of 15, 30 and 60 mg/kg for nine weeks. Results showed that Cd could dose-dependently deposited in magnum and a positive correlation occurred between the accumulation of Cd in magnum and Cd content in egg whites. When compared with control group, 60 mg/kg Cd decreased magnum ovomucin, ovalbumin, lysozyme and ovomucoid gene expressions, but increased avidin gene expression obviously. Hens receiving 60 mg/kg Cd exhibited a significant decrease in egg white ovalbumin content and its proportion in total protein and increase in total cholesterol and LDLC levels in serum and yolk. The level of GSH and activities of T-SOD and CAT in magnum and GSH-Px activity in egg white were decreased significantly, while MDA level in both magnum and egg white was increased obviously in 60 mg/kg Cd group. Besides, Cd induced oxidative stress, inflammation, endoplasmic reticulum stress (ERS) and mitophagy accompanied by a significant up-regulation of the expressions of Keap1, IL1β, TNFα, NF-κB, p65, p-p65, Grp78, CHOP, ATF4, ATF6, IRE1α, Grp94, PINK1, Parkin, Fundc1, GRP75, VDAC1, MCU, MFF, Bnip3 and LC3I/II and a significant down-regulation of the expressions of Nrf2, HO-1, SOD1, SOD2, SOD3, MFN1, MFN2 and PGC1α. These results indicated that Cd can increase serum and yolk cholesterol level and induce magnum oxidative stress, inflammation, ERS and mitophagy through Nrf2/NF-κB and IRE1α/IP3R/GRP75/VDAC1/MCU pathways, thus disrupting egg white composition. Highlights Cd exposure disrupted the synthesis of egg white protein in oviduct magnum and destroyed antioxidant status and protein composition in egg white. Cd exposure significant enhanced serum and yolk total cholesterol and LDL cholesterol levels. Cd exposure induced magnum oxidative stress, inflammation, ERS and mitophagy through Nrf2/NF-κB and IRE1α/IP3R/GRP75/VDAC1/MCU pathways.

MM-210 Targeting Proteasome-Dependent Antigen Presentation to Enhance T Cell–Mediated Tumor Killing

Cancer immunotherapy has emerged as a leading strategy in the war on cancer; however, only a minority of patients exhibit maximal benefits and durable responses. Proteasomes are central components of the proteolytic processing machinery required to generate antigenic peptides presented by MHC class I molecules to cytotoxic T lymphocytes (CTLs). Since cancer cells downregulate the generation and presentation of antigenic peptides to escape anti-tumor immunity, we hypothesized that proteasomal generation of antigenic peptides could be pharmacologically enhanced to increase tumor antigen presentation and enhance CTL-mediated tumor lysis. We performed a cell-based, high-throughput screen (HTS) using ~3,400 FDA-approved drugs and bioactive molecules to identify agents that increased proteasomal peptide-hydrolyzing activity. We then determined whether "hits" identified in the HTS also increased the presentation of a specific MHC class I antigen, SIINFEKL, which is derived from chicken ovalbumin. We engineered a T cell that specifically expresses a T-cell receptor (TCR) that recognizes SIINFEKL. Tumor cells were pre-treated with hits identified in the HTS, most notably the histone deacetylase (HDAC6) inhibitors Tubastatin A and ACY-1215, and then incubated with the engineered T cells to determine the effect on T cell-mediated tumor lysis. We found that Tubastatin A and ACY-1215 potently increased proteasome activity in the cell-based assay. We employed the lymphoma cell line EG.7-ova that constitutively expresses the chicken ovalbumin gene. Proteasomal degradation of ovalbumin generates a peptide, SIINFEKL, that is presented at the tumor surface complexed with the MHC class I molecule. Treatment of EG. 7-ova cells with HDAC6 inhibitors enhanced the presentation of the SIINFEKL:MHC class I complex nearly 3-fold. CTLs were then engineered to express a TCR that specifically recognizes SIINFEKL. When EG.7-ova cells were pre-treated with HDAC6 inhibitors and then co-cultured with SIINFEKL-restricted CTLs, tumor lysis was also increased up to 3-fold. Western blotting showed that treatment of MM cells with HDAC6 inhibitors increased immunoproteasome levels and catalytic activity. Taken together, our results endorse a paradigm-shifting approach to exploit pharmacologics that enhance the proteasomal generation of tumor antigens as an anti-myeloma strategy. Pharmacologic modulation of proteasome activity potently stimulates the presentation of tumor antigens and represents an actionable approach to trigger anti-myeloma immunity.

Abstract 2111: Mobilization of memory natural killer cells in cancer immunotherapy

Abstract Introduction: Immunological memory has long been exclusively attributed to T and B lymphocytes; however, we now know that natural killer (NK) cells also possess an analogous function. Studies of Rag-1-deficient mice (Rag1-/-), which lack T and B cells, provided evidence of NK cell-mediated immunological memory. As NK cells also possess a natural capacity to eliminate tumor cells, we set out to define the role of NK cell memory in anti-cancer immune responses using NK cell-targeted cancer immunotherapy through immunization. Methods: Rag1-/- mice (n=15) were immunized with the E749-57 RAHNIVYTIF (R9F) peptide from Human Papillomavirus 16, using the proprietary DepoVax (DPX) vaccine formulation, (IMV, Inc., NS Canada). Age-matched Rag1-/- mice (n=15) treated with the DPX vehicle alone served as controls. Sixteen days after immunization, all mice were flank-injected with C3 tumor cells, which express the R9F antigen. Tumor appearance and tumor growth rates were recorded. To test the requirement for Perforin, Rag1/Prf1/- mice (n=15) or age-matched Rag1-/- (n=15) were similarly immunized and tumor challenge. To determine the antigen specificity of NK cell memory, Rag1-/- mice (n=15) were immunized with the chicken-ovalbumin model antigen OVA257-265 SIINFEKL (DPX-SIINFEKL) or DPX vehicle (n=15). Mice for these experiments were implanted with C3 cells that express the ovalbumin gene and present the SIINFEKL antigen (C3-OVA cells). For all groups, tumor appearance and tumor growth rates were recorded. Results: Rag1-/- mice vaccinated with DPX-R9F had better protection against C3 tumor development, with 60% remaining tumor-free in comparison to the DPX vehicle control cohort, in which all mice developed tumors. Additionally, the tumor growth rate in DPX-R9F immunized mice was significantly slower than in the control cohort. Perforin has a key role in mediating the observed anti-tumor responses as Rag1/Prf1/- mice vaccinated with DPX-R9F had less tumor protection compared to the Rag1-/- mice, of which only 20% remained tumor-free. Finally, we found 60% of mice immunized with DPX-SIINFEKL and challenged with C3-OVA remained tumor-free, indicating that tumor protection could be induced by distinct antigens. Conclusions: Our preliminary results suggest that DPX-R9F or DPX-SIINFEKL immunization induces antigen-specific protection against tumor development in mice in a T cell- and B cell-independent manner. These immunizations appear to induce memory NK cells to mount antigen-specific antitumor responses that rely on Perforin as an effector molecule. By demonstrating that memory NK cells have a role in protection against tumor development, future cancer immunotherapies could be improved by priming not only T cells but also NK cells, proposing a therapeutic approach with better patient outcomes and improving, at the same time, the safety profile of these immunotherapies. Citation Format: Daniel Medina-Luna, Gayani Gamage, Michal Scur, Haggah Zein, Brendon Parsons, Andrew P. Makrigiannis. Mobilization of memory natural killer cells in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2111.

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

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.

Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens.

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4–1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

Deleterious amino acid substitutions with a series of putative damaging effects on egg components are revealed in the ovalbumin gene family; an in silico approach

Abstract This study was conducted to identify the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) in the ovalbumin gene family, including OVALX, OVALY, and OVAL genes, which are involved in the synthesis of the most important components in the chickens’ eggs using a comprehensive in silico approach. Ten different computational servers were utilized to prioritize the possible deleterious effects of the retrieved nsSNPs in terms of structure, function, and stability. Results indicated entirely damaging effects of H365P in OVALX, I167T in OVALY, and V209G, L231P, F307C, and S317P in OVAL proteins. Further prediction tools showed that all of these deleterious nsSNPs were positioned in variable locations within several α-helix motifs in all studied ovalbumin proteins. Furthermore, all witnessed nsSNPs were predicted to be resided in the receptors binding sites, signifying remarkable involvement of such nsSNPs in damaging of the altered proteins. In conclusion, the present study provides the first inclusive data with regard to the most deleterious nsSNPs in OVALX, OVALY and OVAL genes in chickens. The present bioinformatics data may be useful for breeders who intend to raise chickens for egg production, in such a way the presence of any of these deleterious nsSNPs in any selected breed may possess several damaging effects on the egg components, which may impair egg production. Therefore, it can be stated that breeders have to confirm the absence of any of these deleterious nsSNPs before being proceeded further for large-scale egg-production purposes.

Potential of transferring transgenic DNA from silkworm to chicken

Safety assessment must be conducted before the commercial release of transgenic silkworms. This study was conducted to assess the potential of transferring transgenic DNA from silkworms to other organisms. One hundred hatched male chickens were evenly assigned into 4 groups (T1–4). Groups T1–3 were fed transgenic silkworms P3+5UI with enhanced green fluorescent protein DNA (EGFP) inserted, A4SOR with superoxide reductase DNA (SOR) inserted, and normal silkworm, respectively. Each chicken was fed one silkworm larva every day for 3 weeks. T4 was the normal feeding control. Twenty chickens were randomly selected from each treatment for sacrifice at 22 days of age. The serum was collected individually for biochemical examination, revealing no difference in the analyzed serum parameters between T4 and T1–3. DNA from the duodenum, jejunum, ileum, liver, kidney, and jejunal digesta was extracted for PCR analysis of EGFP, SOR, silkworm housekeeping gene TIF-4A, and chicken ovalbumin gene. No transgenic DNA or TIF-4A was detected in the digesta and tissues of chickens. The same results were observed in chicken upon increasing the amount and frequency of feeding transgenic silkworms, suggesting that the transgenic DNA from silkworms was degraded in the digestive tract and not transferred into the tissues of chicken. This study revealed that transferr recombinant DNA from transgenic silkworm to another organism is unlikely.

Genetic evaluation of Central Javanese local duck based on the ovalbumin gene

Duck farm industry has a big contribution to the supply of animal protein sources in Indonesia. Reproductive efficiency in poultry is determined by the factor of the high ratio of hatching and fertilization, fertile duration, egg weight, and the number of eggs. Reproductive efficiency control genes play a major role in parent selection and provision of superior duck seeds. This study aims to evaluate the genetic quality of local ducks in Central Java based on the ovalbumin gene using PCR-RFLP technique. A total of 35 central Javanese local ducks were feathered on the inside of the right and left wings. Duck DNA is isolated from a feather sample. The ovalbumin gene was amplified with PCR techniques using specific primers TovaF1 and TovaR1. The product of the ovalbumin gene amplification (350 bp) is subsequently cut with restriction enzyme SspI (5′-AATATT-3′). DNA bands from restriction enzyme cutting then analyzed its genotype. The results showed that 6(17.14%) ducks had CC genotype, 16(45.71%) with TT genotype, and 13(37.14%) of CT genotype. Ducks with CC and TT genotypes showed higher hatchability compared to CT genotype. It was concluded that 22(62.86%) central Javanese local ducks were genetically superior in terms of hatchability (genotype CC and TT).

Abstract CT119: Trial in progress: First in human Phase I study using a plasmid DNA coding for Emm55 streptococcal antigen (IFx-Hu2.0) in patients with unresectable stage III or stage IV cutaneous melanoma

Abstract Background: In the past few years, anti-PD-1 and anti-CTLA4 immune-based therapeutics have revolutionized melanoma therapy, but many patients do not respond. The IFX-Hu2.0 formulation consists of a vector encoding a streptococcal membrane protein Emm55 and a cationic polymer. When IFX-Hu2.0 is injected into murine or equine melanomas, tumors regress. Transfection of the emm55 vector into B16 melanoma cells resulted in expression of Emm55 as measured by RT-PCR. Systemic immune responses elicited by IFx-Hu2.0 in B16 bearing mice were CD4 and CD8 T cell dependent and antigen specific. Antigen specificity was tested utilizing a M05 tumor model (B16 cells transfected with the Ovalbumin gene). Mice received 5*106 OT-1 T cells (T cells that have transgenic TCR that recognizes OVA). Tumors were harvested (48 hours) and tetramer specific CD8+ T cells were increased in treated mice. Given this preliminary data, we proceeded to design a first in human Phase I feasibility trial (single dose level with no dose escalation) to test IFx-Hu2.0 use as an intralesional immunotherapy for melanoma to provoke the immune response by facilitating Emm55 antigen surface expression and enhancing tumor recognition. Methods: The primary objective is to assess the safety, tolerability, and feasibility of intralesional injection with IFx-Hu2.0 as a monotherapy in 6 adult patients (&amp;gt;18 years) with unresectable stage III or stage IV cutaneous melanoma lesions. To be eligible, a patient must have at least 1 injectable lesion and 1 un-injected lesion available for biopsies, but up to 3 lesions may be injected. Unlike other trials for melanoma, only 3 mm cutaneous lesions are needed. Patients must have failed, refused or been deemed not candidates for one form of systemic anti-PD-1-based immunotherapy as well as BRAF inhibition, if BRAF V600 mutated. In addition, patients with unresectable cutaneous, subcutaneous, and nodal melanoma lesions recurrent after initial surgery must have failed, refused or not candidates for TVEC. Patients may be enrolled with brain metastases (&amp;lt;1 cm) as long as they are receiving radiation with a life expectancy of &amp;gt;3 months, but may not be receiving concurrent chemotherapy/biological therapy, have uncontrolled hepatitis B/C/HIV infection, or have a history of organ allograft transplantation. Concurrent radiotherapy is allowed at distant sites from the injected lesion. 40 mL of peripheral blood will be collected prior to treatment and at the follow-up visit (28 days) for correlative studies to complement the preclinical murine models. Patients responding to therapy may continue dosing every 3 weeks thereafter. Feasibility is defined as the ability to treat five of the six patients enrolled without dose-limiting toxicity (28 days). As of January 2019, 1/6 of the planned patients have been enrolled. Clinical trial registry number: NCT03655756. Citation Format: Joseph Markowitz, Krithika N. Kodumudi, Deanryan B. De Aquino, Vernon K. Sondak, Shari Pilon-Thomas. Trial in progress: First in human Phase I study using a plasmid DNA coding for Emm55 streptococcal antigen (IFx-Hu2.0) in patients with unresectable stage III or stage IV cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT119.

Polysaccharide from Fruits of Physalis Alkekengi L. Enhances Antitumor Efficacy by a DNA Vaccine

Physalis. alkekengi fruit has long been used in traditional Chinese medicine for tumor therapy. In the present study, using plasmids that encode ovalbumin (OVA) gene, we investigate the adjuvant activity of a polysaccharide fraction (PPSB) isolated from P.alkekengi fruit. Formulation by simple procedures of mixing of the OVA-encoding pCI-neo-sOVA plasmid with PPSB not only induced specific antibody responses, but also induced antigen-specific cytotoxic T lymphocyte (CTL) responses (Graph abstract). Furthermore, immunization using this vaccine prevented the growth of OVA-expressing B16-OVA tumor cell growth in the immunized mice. Thus, we provide evidence supporting the adjuvant activity of PPSB in DNA vaccine against tumor.

Efficient production of human interferon beta in the white of eggs from ovalbumin gene\u2013targeted hens

Transgenic chickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenic chickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-β) into the chicken ovalbumin locus and producing hIFN-β in egg white. We knocked in hIFN-β into primordial germ cells using a CRISPR/Cas9 protocol and then generated germline chimeric roosters by cell transplantation into recipient embryos. Two generation-zero founder roosters produced hIFN-β knock-in offspring, and all knock-in female offspring produced abundant egg-white hIFN-β (~3.5 mg/ml). Although female offspring of the first generation were sterile, their male counterparts were fertile and produced a second generation of knock-in hens, for which egg-white hIFN-β production was comparable with that of the first generation. The hIFN-β bioactivity represented only ~5% of total egg-white hIFN-β, but unfolding and refolding of hIFN-β in the egg white fully recovered the bioactivity. These results suggest that transgene insertion at the chicken ovalbumin locus can result in abundant and stable expression of an exogenous protein deposited into egg white and should be amenable to industrial applications.

Effects of dietary methionine on productivity, reproductive performance, antioxidant capacity, ovalbumin and antioxidant-related gene expression in laying duck breeders.

The study investigated whether dietary methionine (Met) affects egg weight and antioxidant status through regulating gene expression of ovalbumin (OVAL), nuclear factor erythroid 2 like 2 (Nrf2) and haem oxygenase 1 (HO-1) in laying duck breeders. Longyan duck breeders (n 540, 19 weeks) were randomly assigned to six treatments with six replicates of fifteen birds each. Breeders were fed diets with six Met levels (2·00, 2·75, 3·50, 4·25, 5·00 and 5·75 g/kg) for 24 weeks. The egg weight (g), egg mass (g/d), feed conversion ratio, hatchability, 1-d duckling weight, albumen weight, albumen proportion and OVAL mRNA level improved with dietary Met levels, whereas yolk proportion decreased (P<0·05). The weight of total large yellow follicles increased linearly (P<0·001) and quadratically (P<0·05) with dietary Met concentration, and their weight relative to ovarian weight showed a linear (P<0·05) effect. Dietary Met level had a linear (P<0·05) and quadratic (P<0·001) effect on the gene expression of glutathione peroxidase (GPX1), HO-1 and Nrf2, and quadratically (P<0·05) increased contents of GPX and total antioxidant capacity (T-AOC) in liver of duck breeders. In addition, maternal dietary Met enhanced gene expression of GPX1, HO-1 and Nrf2, increased contents of GPX and T-AOC and reduced carbonylated protein in the brains of hatchlings. Overall, dietary Met concentration affected egg weight and albumen weight in laying duck breeders, which was partly due to gene expression of OVAL in oviduct magnum. A diet containing 4·0 g Met/kg would achieve optimal hepatic GPX1 and Nrf2 expression, maximise the activity of GPX and minimise lipid peroxidation.

Genetic fusion protein 3×STa-ovalbumin is an effective coating antigen in ELISA to titrate anti-STa antibodies.

Heat-stable toxin type I (STa)-ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti-STa antibodies for ETEC vaccine candidates. STa-ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti-STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa-ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti-STa antibody titration ELISA. Data showed fusion protein 3×STa-ovalbumin was effectively expressed and extracted, and anti-STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa-ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti-STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa-ovalbumin is an effective ELISA coating antigen for anti-STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

Identification of Egg White Proteins and Divergence in the Regulatory Region of the Ovalbumin Gene in Avians

Egg white proteins play an important role in avian reproductive systems and are an ideal resource for bioreactor construction. In this study, 1D electrophoresis and MALDI-TOF-MS were performed to analyze egg white proteins in four species. In total, 18, 11, 28, and 13 proteins were identified in the egg whites of the chicken, duck, goose, and pigeon, respectively. Egg white proteins in chickens have been studied previously; therefore, we focused on the proteins in goose and duck egg whites. Based on the amino acid sequence analysis and a comparison of the unique peptides, high similarity was observed between the goose and duck egg whites. Diversity in the regulatory region of the ovalbumin gene explained the higher ovalbumin expression in the duck and goose than in the chicken. These data clarify the evolutionary processes underlying to the unique peptides contributing to the differential expression of ovalbumin in avians.

Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System.

In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.

The Family Secrets of Avian Egg-Specific Ovalbumin and Its Related Proteins Y and X.

The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control, but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX, and OVAY belong to the serine protease inhibitor (serpin) family whose members share a common tertiary structure. Ovalbumin and OVAX are one of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molecule. Ovalbumin is the major egg white protein and might be a source of amino acids for the developing embryo. The physiological function of OVAY is not known, but recent data have revealed a possible role of this protein in early embryonic development. Considering the antibacterial activities of OVAX, this protein might play a role in egg defense. This review sheds light on the expression, biochemistry, and structural specificities of these three highly similar paralogs. It gives new clues in favor of diverging functions, which are likely to have arisen by duplication events from a common ancestral gene.