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.
Highlights
Pigs are an attractive research model in the field of surgical and procedure training, disease progression and pathology, translational research, and regenerative medicine/stem cell therapy, due to their anatomic, genetic, and physiological similarities with humans
To determine whether the effect was somatic cells nuclear transfer (SCNT)-related, cauda epididymal sperm were isolated from a Model I boar, frozen and used for in vitro fertilization (IVF) with wild type oocytes obtained from the slaughterhouse
A total of 212 zygotes obtained via IVF were transferred to a recipient sow, resulting in the birth of three piglets, with two piglets expressing pH2BeGFP (Model I, F1 generation)
Summary
Pigs are an attractive research model in the field of surgical and procedure training, disease progression and pathology, translational research, and regenerative medicine/stem cell therapy, due to their anatomic, genetic, and physiological similarities with humans. Genetic engineering of encoded biological fluorescent proteins have marked an evolution in the field of stem cell biology, allowing the development of cell-traceable systems and the ability to track the fate of adult stem cells for therapeutic purpose in biomedical models [9, 10]. Among these molecular tags, the most widely used one is the green fluorescent protein (GFP) from the jellyfish Aequorea victoria [6, 11]. The mutant variant, eGFP (enhanced), has been shown to be more stable and to fold correctly at 37 ̊C, and can be fused with virtually any protein of interest allowing co-localization of eGFP and the protein of interest. [6]
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