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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. [ABSTRACT FROM AUTHOR]

Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in preand postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 μm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P < 0.05. We determined mRNA expression for kisspeptin was not different between groups (P > 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P < 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P < 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P < 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin. [ABSTRACT FROM AUTHOR]

Puberty onset in gilts is an awakening of the hypothalamic-pituitary-ovarian axis that is the result of reduced estradiol-negative feedback at the level of the hypothalamus which yields elevated gonadotropin secretion from the anterior pituitary. Given the importance of hypothalamic kisspeptin and neurokinin B (NKB) signaling for the onset of puberty in other species, the objective of this study was to determine if gilts selected for early pubertal onset (SELECT) would display measurable differences within the hypothalamus (i.e. increased expression of kisspeptin and NKB) and within the ovary (i.e. increased ovarian mass) compared to age-matched and weight-matched gilts (CONTROL) that achieve puberty 20 days later than SELECT gilts. Gilts were sacrificed at three timepoints: Timepoint A, both groups were determined to be prepubertal (n=6/group), Timepoint B, SELECT gilts were determined to be pubertal and CONTROL gilts were determined to be prepubertal (n=6/group), and Timepoint C, both groups were determined to be pubertal (n=6/group). All animals were euthanized, heads were perfused with 8 L of 4% paraformaldehyde, and ovaries were harvested. Brain tissue was removed post-fixation, submerged in fixative for 24 hrs followed by 20% sucrose until sectioned for immunohistochemistry. Ovarian mass tended (p=0.10) to be greater for SELECT gilts on the right ovary (4.34 vs. 3.67 g) and the left ovary (4.49 vs. 3.68 g) when compared to CONTROL (Timepoints A and C), and at Timepoint B right ovary mass from SELECT gilts was heavier than CONTROL gilts (p< 0.05; 7.22 vs. 4.65 g). Hypothalamic immunohistochemistry for kisspeptin and NKB revealed differences in neuronal fiber density between both groups at various timepoints. Therefore, we conclude that gilts genetically selected for early puberty do so via changes within the hypothalamus that increase gonadotropin secretion and, in turn, stimulate ovarian growth to ultimately advance the timing of puberty onset. [ABSTRACT FROM AUTHOR]