Location: Plant Genetics Research
2022 Annual Report
Objectives
Objective 1: Using gene editing techniques, systematically alter DNA sequence predicted to cause beneficial changes in pig production and test these changes in vitro and in vivo where warranted.
Objective 2: Comprehensively test effects of sequence modifications for on and off target effects.
Objective 3: Develop efficient methods for gene modification of poultry.
Approach
Using gene editing techniques, we will systematically alter DNA sequence predicted to cause beneficial changes in pig production and test these changes in vitro and in vivo where warranted. In addition, we will comprehensively test effects of sequence modifications for on and off target effects. Gene editing and other technologies will be used to systematically alter DNA sequence in ways that were predicted to improve traits to (1) determine whether the sequence variation is causal for the change in the trait, and (2) determine any other changes in traits that might simultaneously occur through pleiotropy effects. The research will focus on pigs, and the goal will be to develop pigs with improved traits of interest to the swine industry, without having deleterious effects on swine production. Simultaneously, we will work toward understanding relationships between genes and various physiological functions within pigs. This information may also be valuable to both humans and other livestock species.
Progress Report
Objective 1. ARS researchers in Columbia, Missouri, have established multiple porcine cell lines including immortalized porcine muscle satellite cells, primary myocytes, and primary adipocytes. Currently, researchers are working to establish an immortalized porcine kidney cell line, an established hepatocyte and/or bile duct cell line, an immortalized pulmonary alveolar macrophage cell line and intestinal organoids.
In collaboration with ARS researchers in Clay Center, Nebraska, we have identified potential causative variants associated with age at puberty. Two variants are being targeted currently. The guides and assays have been designed and the modifications are in progress. Other candidate genes have been identified and assays are currently being developed.
Objective 2. Over the past year, ARS researchers and collaborators in Columbia, Missouri, have made progress exploring the incidence of off-site targeting in gene edited pigs. Whole genome sequencing has been completed on tissues from pigs that have been created by using guides that have a high incidence of off-site targeting, pigs produced by guides that have low incidence of off-site targeting, and a wild-type control to determine levels and regions where off-site targeting occurred. Sequence has been generated and aligned to the Sus scrofa 11.1 reference genome with a high coverage of reads. Off-site or unwanted targets have been identified and are currently being analyzed.
Off-site targeting is currently being analyzed in embryos injected with guides that have a high incidence of unwanted modifications and RNA encoding anti-clustered regularly interspaced short palindromic repeats (CRISPR) proteins. These embryos are being analyzed to determine if the anti-CRISPR protein decreases unwanted modifications and is ongoing.
Objective 3. ARS researchers in Columbia, Missouri, have established chicken embryo fibroblast cells and B cells that were obtained from a commercial source. These have been propagated and cryopreserved. Other poultry cell lines are being established now. Quail and chicken breeding flocks are being established. ARS researchers in Columbia, Missouri, have formed collaborations with ARS researchers in East Lansing, MI and Athens, GA and have identified gene targets for producing disease resistant poultry. Genetic targets have been designed, and transfections are being performed in cell lines.
Accomplishments
Review Publications
Lucas, C.G., Redel, B.K., Chen, P.R., Spate, L.D., Prather, R.S., Wells, K.D. 2021. Effects of RAD51-stimulatory compound 1 (RS-1) and its vehicle, DMSO, on pig embryo culture. Reproductive Toxicology. 105:44-52. https://doi.org/10.1016/j.reprotox.2021.08.002.
Chen, P., Redel, B.K., Kerns, K., Spate, L.D., Prather, R.S. 2021. Challenges and considerations during in vitro production of porcine embryos. Cells. 10(10):2770. https://doi.org/10.3390/cells10102770.
Chen, P.R., Uh, K., Redel, B.K., Reese, E., Prather, R.S., Lee, K. 2022. Production of pigs from porcine embryos generated in vitro. Frontiers in Animal Science. 3. Article 826324. https://doi.org/10.3389/fanim.2022.826324.
