Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Our target audience includes researchers in animal science, developmental biology, poultry science, biotechnology, and industry. The new insights gained from this project will contribute to scientific knowledge for education and offer potential applications in animal production and the food industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided valuable training opportunities for a diverse team, including one graduate student, one postdoc, one technician, and one undergraduate student, equipping them with essential in vitro and in vivo research skills. Their in vitro training included plasmid construction for target guide RNA and Cas9 gene, cell culture, gene overexpression in cultured cells, and microscopic image analysis. In vivo techniques covered in ovo injection, embryonic fat tissue dissection, histological analysis, quail breeding, and statistical data analysis. Beyond hands-on experience, this project deepened their understanding of developmental biology, molecular biology, cell biology,nutrition, and poultry science, fostering a comprehensive skill set for future scientific endeavors. How have the results been disseminated to communities of interest?To share our findings, we are preparing manuscripts for peer-reviewed journals in animal and poultry sciences, ensuring broad accessibility and impact. Students and a postdoc have presented key results at local conferences, including the Russell Klein Nutrition Research Symposium and the College of Food, Agricultural, and Environmental Sciences Research Forum at The Ohio State University, gaining valuable experience and feedback. Additionally, the PD has expanded our research visibility through invited seminars at national and international conferences, universities, and research institutes, fostering collaboration and knowledge exchange. These efforts ensure our findings reach diverse audiences and contribute meaningfully to the field. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we will continue our efforts to generate genome-edited poultry targeting the RBP7 gene and examine the effects of RBP7 inactivation on adipose development in vivo. Additionally, we will explore the impact of in ovo retinoic acid injection on intramuscular fat development in chicken embryos. Further, we will investigate how dietary vitamin A levels influence fat accretion and key adiposity parameters, including total fat cell size and number, in post-hatch chickens. These studies will provide deeper insights into the regulatory mechanisms of fat deposition in poultry.
Impacts
What was accomplished under these goals?
In 2024, we made significant progress in generating genome-edited poultry targeting the RBP7 gene and exploring its role in adipose development. We successfully tested two guide RNAs for gene editing in quail cells, leading to the injection of 517 fertile eggs with adenovirus carrying both gRNAs. This resulted in the successful hatching of 31 potential germline chimeras. Hundreds of their offspring have been screened using PCR and sequencing, with additional generations continuously being produced and analyzed to establish gene-edited quail. To further enhance efficiency, we identified a highly effective gRNA in cultured quail cells, and recombinant adenoviruses carrying this optimized gRNA are now being injected into quail blastoderms. These efforts are steadily advancing the development of RBP7-edited quail, bringing us closer to achieving our research goals. Our in vitro studies on the effects of retinoic acid in adipocyte development revealed that RBP7 is highly expressed in adipocytes, where it promotes adipogenesis by enhancing PPAR-gamma signaling and regulating retinol metabolism. This underscores its potential as a key target for fat regulation in animals. Additionally, we discovered that all-trans retinoic acid (atRA) induces the transdifferentiation of avian myoblasts into adipocytes by directly regulating PPAR-gamma, suggesting its promising role in improving marbling in poultry. These exciting findings have driven us to further explore the role of atRA in myocyte-to-adipocyte transdifferentiation in vivo, with a long-term vision of enhancing marbling. To refine our approach, we are developing strategies to assess the effects of different injection times and dosages of atRA during embryonic development on adipocyte formation in both adipose and muscle tissues. To improve visualization of adipocyte development in whole embryonic tissues, we have also established an innovative KOH-based clearing method that renders muscle tissue transparent while preserving adipose tissue, allowing us to observe fat cell development within muscle tissue more effectively.
Publications
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, biotechnology, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the food animal industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through the project, 1 postdoc, 1 technician, 2 undergraduate students, and 1 visiting scholar were trained to learn in vitro and in vivo techniques. In vitro techniques they learned included construction of plasmids containing target genes, cell culture, overexpression of target genes in cultured cells, and analyses of microscopic images. In vivo techniques include microinjection of vectors into blastoderm, hatching and rearing chicks, breeding colonies of quail, DNA extraction from feathers, PCR, and genotyping. The project provided opportunities for the researchers to learn developmental biology, CRISPR technology, molecular genetics, reproduction, and poultry science. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the project were published in scientific journals to be available to the scientific community in animal and poultry sciences. Students and a postdoc presented the results from the project in the national meeting including the Poultry Science Association (PSA) Annual Meeting, and local meetings including the Russell Klein Nutrition Research Symposium, and the College of Food, Agricultural, and Environmental Sciences Research Forum at The Ohio State University. PD also presented in invited seminars for several universities and research institutes. What do you plan to do during the next reporting period to accomplish the goals?For the 3rd year of the project, we will continue generation of genome-edited poultry in RBP7 gene and study the effects of RBP7 inactivation on adipose development in vivo. In addition, we will investigate the effect of dietary vitamin A concentrations on fat accretion and adiposity parameters including total fat cell size and number in post-hatch chickens.
Impacts
What was accomplished under these goals?
For the 2nd year, we have been focusing on generating genome-edited quail line and planning for in vivo study investigating effects of dietary vitamin A concentrations on growth and development of adipose tissue. To avoid designing guide RNAs on any potential variable sequences (sequence polymorphism) of the RBP7 target gene, genomic DNA from quail myogenic (QM) cells and individual quail were isolated and the RBP7 gene was amplified by PCR for DNA sequencing. The sequencing results showed that there are several variable nucleotides in the entire RBP7 coding sequences of quail population that we have at the Ohio State University. Several guide RNAs were selected based on high on-target and low off-target scores using the bioinformatics program (https://www.benchling.com/crispr/). To insert selected gRNA, each pair of oligos for each targeting sequence were annealed with the CRISPR/Cas9 vector. The final constructed targeting vectors which contain targeting guide sequences were confirmed by Sanger sequencing at The Ohio State University DNA Sequencing Core Facility. To verify the targeting efficiency, each of the constructed GFP-tagged vectors were transfected into QM7 cells. After sorting of GFP positive cells, the pooled GFP positive cells were subjected to DNA extraction and followed by PCR amplification of genomic DNA containing the potential target sites. Among several guide RNAs, two guide RNAs have genome editing activities in quail cells. Two different recombinant adenoviruses (>1 × 10^10/mL) containing either the selected guide RNA 1 or 2 were produced. Two different recombinant adenoviruses were injected into the central area of the quail blastoderm via our microinjection system. A total of 317 eggs were injectedwith the first adenoviruses (2 μL) containing gRNA1 and rotated automatically in the incubator. Among 17 hatchlings, 15 potential germ-line chimeric quail survived and maintained. These chimeric founder lines were currently mated with wild-type (WT) quail to produce offspring that have screened for gene-edition in the RBP7 locus. Unfortunately, there were no offspring with gene-edition in the RBP7 locus as screened by PCR followed by sequencing the PCR products. The second adenoviruses containing gRNA2 were injected into blastoderm of 200 fertile eggs. A total of 21 hatchlings were sexually matured and mated. Offspring from breeding of these potential chimeric quail are now being screened by PCR followed by sequencing the PCR products.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kim DH, Lee J, Lee C, Shin BJ, Ryu BY, Lee K., In ovo injection of all-trans retinoic acid causes adipocyte hypertrophy in
embryos but lost its effect in posthatch chickens. Animal. 17(4):100750
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lee, B., Kim, D.-H., Lee, J., Cressman, M. D., Choi, Y. M., and Lee, K. (2023). Greater numbers and sizes of muscle bundles in the breast and leg muscles of broilers compared to layer chickens. Front. Physiol. 14, 1285938. doi:10.3389/fphys.2023.1285938.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Target audiences are researchers in the area of animal science, developmental biology, poultry science, biotechnology, and industry. New information in this project will provide scientific knowledge for education and a potential application for animal production and the food animal industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Through the project, 1 graduate student, 1 postdoc, 1 technician, and 2 undergraduate students were trained to learn in vitro and in vivo techniques. In vitro techniques they learned included construction of plasmids containing target genes, cell culture, overexpression of target genes in cultured cells, analyses of microscopic images, and assays for measuring lipid contents in cells. In vivo techniques include in ovo injection, dissection of embryonic fat tissue, histological analysis, hatching and rearing chicks, and statistical analysis of data. The project provided opportunities to the researchers to learn developmental biology, cell biology, nutrition, and poultry science. How have the results been disseminated to communities of interest?For the dissemination of our findings from the project, we prepared and submitted manuscripts. The results from the project were published in scientific journals to be available to the scientific community in animal and poultry sciences. Students and a postdoc presented the results from the project in the local meetings including the Russell Klein Nutrition Research Symposium, and the College of Food, Agricultural, and Environmental Sciences Research Forum at The Ohio State University, and the virtual NCCC210 reginal meeting (Regulation of Adipose Tissue Accretion in Meat-Producing Animals). PD also presented in invited virtual seminars for several universities and research institutes. What do you plan to do during the next reporting period to accomplish the goals?For the 2nd year of the project, we will continue working on generation of genome-edited poultry in RBP7 gene and study the effects of vitamin A concentrations in diets on growth performance and adiposity parameters including total fat cell number, adipocyte size, and fat accretion in post-hatch chickens.
Impacts
What was accomplished under these goals?
The regulation of adipose deposition in broiler chickens is an important factor for production efficiency to poultry producers and health concerns to customers. Although vitamin A and its metabolite [all-trans retinoic acid (atRA)] have been used for studies on adipogenesis in mammals and avian, effects of embryonic atRA on adipose development in embryonic (E) and posthatch (D) ages in broiler chickens have not been studied yet. Different concentrations of atRA (0 M-2 μM) were injected in broiler eggs at E10, and adipose tissues were sampled at E16. Percentages of adipose tissues in chicken embryos were significantly increased in the group injected with 500 nM of atRA compared to the 0 M group (P < 0.05). In addition, the adipocyte cross-sectional area (CSA) was significantly greater by in ovo injection of 500 nM atRA compared to the injection of 0 M (P < 0.01). Moreover, in ovo atRA-injected embryos were hatched and BWs were measured at D0, D7, and D14. BWs were not different from those of the 0 M group. Percentages of adipose tissues and CSA of the in ovo atRA-injected group (500 nM) were not different from those of the 0 M group at D14. Taken together, the current study clearly showed that in ovo injection of atRA promoted adipose deposition with hypertrophy during embryonic development, but its effects were not maintained in early post-hatch age in broiler chickens, implying that embryonic atRA has an important role in the regulation of adipose development in chicken embryos. Increased adipogenesis in muscle tissues is related to improvement of meat quality in animal production. With growing evidence for pro-adipogenic functions of all-trans-retinoic acid (atRA), the current study investigated whether atRA can transdifferentiate myoblasts into adipocytes using a quail myogenic cell line (QM7) and avian primary myoblasts. AtRA increased cytoplasmic lipid droplet accumulation and mRNA expression for adipogenic genes in these cells. An acute induction of Ppar-gamma expression by atRA under cycloheximide treatment indicated a direct regulation of Ppar-gamma by atRA. In addition, the induction of Ppar-gamma expression was mediated by retinoic acid receptors. At high levels of Ppar-gamma by atRA, BADGE, an antagonist of Ppar-gamma, inhibited, and rosiglitazone, an agonist of Ppar-gamma, further enhanced atRA-induced transdifferentiation. However, at very low levels of Ppar-gamma in the absence of atRA treatment, rosiglitazone could not induce transdifferentiation of avian myoblasts. These data suggest that the induction of Ppar-gamma expression by atRA is an essential molecular event in myoblasts for atRA-induced transdifferentiation into adipocytes. Based on our findings, atRA can be a new transdifferentiation factor of myoblasts to adipocytes, providing a potential nutrient to enhance marbling in poultry. Retinoic acids (RA) are crucial for cell differentiation, including adipogenesis. Retinol binding protein 7 (Rbp7) is under the control of PPAR-gamma, the master regulator of adipogenesis. However, the role of RBP7 in adipogenesis is unclear. Our study showed that Rbp7 was abundantly expressed in white and brown mouse adipose tissues and had a higher expression in adipocytes than in stromal vascular fraction. Rbp7 overexpression promoted 3T3-L1 preadipocyte differentiation with increased triglyceride accumulation and up-regulation of Ppar-gamma, Fabp4, C/ebp-alpha, and AdipoQ. Rbp7 deficient adipocytes had opposite effects of the overexpression, which were rescued by RA supplementation. Indirect assessment of relative nuclear RA levels using RAR response element (RARE)-Luc reporter assay demonstrated that Rbp7 overexpression significantly increased RARE-Luc reporter activity. Rbp7 overexpression significantly increased expression of Raldh1, responsible for RA production, and up-regulation of Lrat and Cyp26a1, involved in retinol storage and RA catabolism, respectively, in 3T3-L1 adipocytes. Rbp7 deficient adipocytes had opposite effects of the overexpression of those genes involved in retinol metabolism. These data suggest that RBP7 increases transcriptional activity of RARE that may induce negative feedback responses via regulation of the gene expression for retinol homeostasis. Our data indicate critical RBP7 functions in adipocytes: regulation of transcriptional activity of RARE and adipocytes differentiation, potentially providing a new target for regulating fat accretion in animals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kim DH, Lee J, Suh Y, Ko JK, Lee K. Transdifferentiation of Myoblasts Into Adipocytes by All-Trans-Retinoic Acid in Avian. Front Cell Dev Biol. 10:856881
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kim DH, Ahn J, Suh Y, Ziouzenkova O, Lee JW, Lee K. Retinol Binding Protein 7 Promotes Adipogenesis in vitro and Regulates Expression of Genes Involved in Retinol Metabolism. Front Cell Dev Biol. 10:876031
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Kim DH, Lee J, Lee C, Shin BJ, Ryu BY, Lee K., In ovo injection of all-trans retinoic acid causes adipocyte hypertrophy in embryos but lost its effect in posthatch chickens. Animal. 17(4):100750
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