title: Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies.
abstract: Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3-5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Renan B
authors.initials: RB
authors.lastname: Sper
authors.affiliation: Department of Surgery and Microbiology and Immunology, University of Michigan Health System, Ann Arbor, Michigan, United States of America.
authors.forename: Sehwon
authors.initials: S
authors.lastname: Koh
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Sean
authors.initials: S
authors.lastname: Simpson
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Animal Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Bruce
authors.initials: B
authors.lastname: Collins
authors.affiliation: Department of Animal Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Jeff
authors.initials: J
authors.lastname: Sommer
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Animal Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Petters
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Caballero
authors.affiliation: Department of Surgery and Microbiology and Immunology, University of Michigan Health System, Ann Arbor, Michigan, United States of America.
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Platt
authors.affiliation: Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Jorge A
authors.identifier: http://orcid.org/0000-0001-9751-3192
authors.initials: JA
authors.lastname: Piedrahita
mesh_terms: D000818:Animals; D058948:Cell Tracking / Q000379:methods*; D033481:Chromosomes, Mammalian / Q000235:genetics / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006657:Histones / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013552:Swine / Q000235:genetics / Q000378:metabolism
chemical_list: D006657:Histones; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins