IDENTIFICATION AND USE OF MAGNUM-SPECIFIC PROMOTERS FOR PRODUCTION OF TARGET PROTEINS IN AVIAN EGGS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017276
• Project Start Date: Apr 17, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Animal Sciences

Non Technical Summary
Animal bioreactors are genetically modified animal systems that have the potential to reduce production costs and improve production efficiency of pharmaceutically relevant recombinant proteins. Several advantages associated with the use of avian as bioreactor systems are 1) Avian species have a short generation time, leading to the rapid establishment of a transgenic line and high egg production, 2) Transgenic avian systems allow for appropriate post-translational modification, as opposed to prokaryotic cell culture bioreactors, and have higher productivity than mammalian cell culture systems, and 3) Recombinant proteins can be incorporated into egg whites and easily collected within the egg. Magnum specific expression of target genes has been achieved by use of the ovalbumin promoter. However, not much real success has been achieved with the ovalbumin promoter, due to the potential leakage of transgene products into circulation that may cause health issues of transgenic birds. Therefore, the main objectives of the hatch proposal are to identify different promoters of magnum specific genes and to validate magnum-specific expression of the GFP reporter gene under the control of the new selected promoters via generation of transgenic quail lines. These new promoters could circumvent current problems associate with leakage of transgene products and could potentially lead to more stable production of transgenic lines.

Animal Health Component

20%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3299	1040	100%

Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology;

Keywords

poultry

bioreactor

transgenic bird

magnum-specific promoter

pharmaceutical protein production

Goals / Objectives
Justification: Animal bioreactors are genetically modified animal systems that have the potential to reduce production costs and improve production efficiency of pharmaceutically relevant recombinant proteins. Several species including goats, cattle, rabbits, and avian have been genetically modified to secrete target proteins into milk, egg whites, blood, or other bodily fluids. There are several advantages associated with the use of avian as bioreactor systems. Avian species have a short generation interval, leading to the rapid establishment of a transgenic line and high egg production. Transgenic avian systems allow for appropriate post-translational modification, as opposed to prokaryotic cell culture bioreactors, and have higher productivity than mammalian cell culture systems. Furthermore, recombinant proteins can be incorporated into egg whites and easily collected within the egg. Magnum specific expression of target genes has been achieved by use of the ovalbumin promoter. However, not much real success has been achieved with the ovalbumin promoter, due to the potential leakage of transgene products into circulation. Therefore, the major goal of the hatch project to identify different promoters of magnum specific genes that could circumvent this problem and could potentially lead to more stable production of transgenic lines.Previous Work and Present Outlook: One of the biggest limitations associated with transgenic animals is gaining appropriate legislation to allow such production systems into the market. In 2009, the first pharmaceutical protein produced by a transgenic animal was approved by the FDA. A biotherapeutics company generated transgenic goats producing antithrombin in their milk via a mammary specific promoter. The second transgenic animal bioreactor system approved by the FDA was a transgenic rabbit model that secretes a drug for the treatment of hereditary angioedema in its milk. Chickens producing human lysosomal acid lipase (Kanuma®) within their eggs were also approved by the FDA in 2015. Currently, these are the only three systems to have gained FDA approval. Despite the low number of approvals, the technology and methods involved in generating transgenic birds for bioreactor systems have been steadily improving over the past few decades.Many advancements have been made in recent decades and several methods for generating transgenic birds have emerged. The most successful and common method is the use of retroviral vectors. Success using retroviral vectors, such as lentiviral vectors, has been well documented. Other methods for the generation of transgenic birds include injection of infected primordial germ cells into the dorsal aorta of recipient embryos and embryonic stem cells into the blastoderm of stage X embryos. It is this constantly advancing technology that is being used in an effort to generate efficient avian bioreactor systems.Since the mid-2000's, a majority of studies have generated transgenic avian bioreactors using retroviral vectors and ubiquitous promoters. Of the 10 studies that used a ubiquitous promoter, six were capable of obtaining generation 1 (G1) transgenic offspring. Quite often, the G1 offspring had phenotypic issues and died at an early age, likely due to the cytotoxicity of transgene products. An option that could potentially decrease the mortality and phenotypic issues of transgenic birds is the use of a magnum-specific promoter to drive the expression of the target gene and production of target protein only in eggs. One such promoter that has been tested previously is the ovalbumin promoter. However, there is a large amount of Ovalbumin within the egg. This high production could potentially lead to considerable leakage even though it is produced in magnum. The use of different promoters of magnum specific genes could improve this situation. Slightly lower production of target proteins within chicken eggs, but without leakiness, may result in proteins that can be purified and used within the pharmaceutical industry, and could potentially lead to more stable production of transgenic lines.The objectives of this Hatch project are; 1) Identification of proteins that are found in eggs by reviewing the literature; 2) Identification of the target genes encoding the egg proteins by analysis of Genbank databases; 3) Evaluation of the target genes to determine if they are exclusively expressed in magnum by means of bioinformatics searches and by reviewing the literature; 4) Confirmation of magnum-specific expression of target genes by real-time PCR; 5) Evaluation of the target genes based on their expression patterns during different reproductive states (i.e., before and after laying eggs); 6) Selection of magnum-specific target genes and identification of their promoters; 7) Cloning of promoters for the selected genes; 8) Generation of transgene constructs containing the promoters + GFP marker gene; 9) Production of viral vectors and microinjection of viral vectors into quail embryos; 10) Validation of transgenic lines for magnum-specific expression of GFP gene by western blot, PCR, and immunohistochemistry analyses.

Project Methods
1) We will search Pubmed to find literature that shows the composition of proteins in egg white. At least 10 proteins that are most abundant in egg white will be selected.2) Genes encoding the 10 proteins will be identified in Genbank. The full length of coding sequences for the 10 proteins will be compiled in a Word document.3) The 10 selected genes will be evaluated to determine whether they are predominantly expressed in magnum by means of a literature review and analysis of microarray databases, if available in the NCBI GEO database.4-5) Selected candidate genes will be investigated for magnum-specific expression by quantitative PCR analysis for avian species including chicken and quail. The cDNA samples for various tissues from these animals were already banked in our laboratory. In addition, the expression patterns of the 10 genes during different reproductive states (i.e., before and after laying eggs) will be investigated by quantitative PCR analysis.6) The promoters of the selected target genes for avian species including chicken, turkey, quail, and Finch will be identified and downloaded to the Word file. Using comparative analysis tools, conserved promoter areas will be identified and transcription factors in the promoters will be compared among avian species. This information will be critical in deciding the sizes of promoters to be used for the transgene constructs.7) The specific primer sets will be designed to amplify target sizes of promoters by PCR reaction using chicken and quail gDNA as templates. The PCR products will be cloned into a pCR2.1 vector and the cloned promoters will be sequenced by The Ohio State University Genome Core Facility.8) Several constructs will be made to contain each promoter and GFP reporter gene.9) The procedures for production, purification, and concentration of lentiviral vectors and microinjection of the viral vectors have been routinely performed in the PD's laboratory as described in previous papers.10) Validation of transgenic lines for magnum-specific expression of the GFP gene will be performed by western blot, PCR, and immunohistochemistry analyses. These procedures have been described in the PD's previous publications.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Target audiences will be researchers in the areas of animal science, poultry science, and pharmaceutical companies. New information in this project will provide scientific knowledge for education and possible applications for poultry science and the pharmaceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, postdoc researchers, a graduate student, and a laboratory technician were trained to perform new assays and tasks. The assay skills they learned in this year include obtaining sequences of the target genes from Genbank, designing primers to amplify target promoters by PCR, cloning of these genes into viral vectors, producing lentivirus containing these genes, and confirming production of GFP in avian cells by infection with the recombinant viruses. The project provided opportunities for the researchers to learn bioinformatics, genetics, molecular biology, and cell biology. In addition, they now grasp the importance of integration of multiple research areas in advancing our knowledge toward the proposed project. How have the results been disseminated to communities of interest?Although we had expected progress in this year, the direct results are too premature to be published. Instead, we have reported several papers that are indirectly related to this project. What do you plan to do during the next reporting period to accomplish the goals?As the Hatch proposal is at the beginning of the 3nd year, we will complete the three objectives: 1) Microinjection of viral vectors into quail embryos, 2) Breeding of founder lines of quail to generate offspring, and 3) Confirmation of transgene integration into offspring genome by PCR analysis.

Impacts
What was accomplished under these goals? During the 2nd year, we have focused on four objectives: 1) Selection of magnum-specific target genes and identification of their promoters, 2) Cloning of promoters for the selected genes, 3) Generation of three transgene constructs containing the promoters + GFP marker gene, and 4) Production of three lentiviral vectors containing the promoters + GFP genes. These four objectives were accomplished in this year as we identified several magnum-specific genes in avian species. To validate expression of viral vectors in avian cells, quail muscle cells were infected with the viral vectors and GFP expression from the viral vectors was confirmed by fluorescence microscope.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahn J, Wu H, Lee J, Hwang IS, Yu D, Ahn JS, Lee JW, Hwang S, Lee K. 2020. The Landscape of Genomic Imprinting at the Porcine SGCE/ PEG10 Locus from Methylome and Transcriptome of Parthenogenetic Embryos. G3: Genes, Genomes, Genetics. 120:401425.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahn J, Wu H, Lee K. 2020. Integrative Analysis Revealing Human Heart-Specific Genes and Consolidating Heart-Related GWAS Loci. Frontiers in Genetics 11:777.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kim DH, Lee J, Suh Y, Cressman M, Lee SS, Lee K. 2020. Adipogenic and Myogenic Potentials of Chicken Embryonic Fibroblasts in vitro: Combination of Fatty Acids and Insulin Induces Adipogenesis. Lipids 55(2):163-171.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahn J, Wu H, Lee J, Hwang IS, Yu D, Ahn J, Lee JW, Hwang S, Lee K. 2020. Identification of a novel imprinted transcript in the porcine GNAS complex locus using methylome and transcriptome of parthernogenetic fetuses. Genes 11(1). pii: E96.

Progress 04/17/19 to 09/30/19

Outputs
Target Audience:Target audiences will be researchers in the areas of animal science, poultry science, and pharmaceutical companies. New information in this project will provide scientific knowledge for student education by providing internships andexperiential learning opportunities. This project focusing development of vector system to produce phamacutical important proteins in chicken eggs through genetic engineering will provide possible applications for poultry and the pharmaceutical industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the project, postdoc researchers (Jinsoo Ahn), graduate students (Rachel Woodfint, Joonbum Lee), a laboratory technician (Yeunsu Suh), and undergraduate students (Erin Hamlin, Jonah Perkins) were trained to perform new assay skills and tasks. The assay skills they learned in this year include literature search, data mining of the Gene Expression Omnibus (GEO) database, and obtaining sequences of the target genes from Genbank. The project provided opportunities for the researchers to learn bioinformatics, genetics, genomics, reproductive biology and molecular biology. In addition, they now grasp the importance of integration of multiple research areas in advancing our knowledge toward the proposed project. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we published one review article entitled "Avian Bioreactor Systems". This review article was also included in the thesis to be available to the scientific community in the area of animal and poultry sciences. What do you plan to do during the next reporting period to accomplish the goals?As the Hatch proposal is at the beginning of the 2nd year, we will complete the next objectives: 4) Confirmation of magnum-specific expression of target genes by real-time PCR, 5) Evaluation of the target genes based on the expression patterns in different reproductive statuses (before and after laying eggs), 6) Selection of magnum-specific target genes and identification of their promoters, 7) Cloning of promoters for the selected genes, and 8) Generation of transgene constructs containing the promoters + GFP marker gene. Through these objectives, we will validate each gene component and generate transgene constructs by putting the gene components together.

Impacts
What was accomplished under these goals? Since beginning this project in August 2019, we have focused on the first three objectives: 1) reviewing the literature to identify proteins that are found in eggs, 2) Identification of the target genes encoding the egg proteins by analysis of Genbank databases, 3) Evaluation of the target genes to determine if they are exclusively expressed in magnum by bioinformatics searches and literature reviews. These three objectives were accomplished in this year as we identified several magnum-specific genes in avian species. To confirm magnum-specific expression of these genes at the mRNA level, five parts of the laying hen's reproductive track, including ovary, infundibulum, magnum, isthmus, and shell gland, were collected. In addition, specific primers for the target genes were designed for quantitative real-time PCR analysis. Through these accomplishments, several proteins that are abundant in eggs were identified. The genes encoding the egg proteins were identified and validated to be expressed in oviduct of hens. This information will be used to obtain the oviduct promoters that will guide production of target proteins in eggs.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Woodfint R, Hanmlin E, Lee K. Avian Bioreactor Systems: A Review. Molecular Biotechnology 60(12):975-983