Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

Abstract

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA−) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA− cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA− hFUT2×hGLA cells as compared to the TSA− nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

“…β-Actin was used as a reference protein (mouse monoclonal IgG designated as anti-β-actin, diluted 1:2000 in TBST; ab8224, Abcam). Afterwards, membranes were washed several times in TBST followed by 1-h incubation with appropriate HRP-conjugated secondary antibodies (i.e., goat anti-rabbit IgGs against human α1,2-FT and goat anti-mouse IgGs against β-actin; Thermo Fisher Scientific), each at a dilution of 1:6000 in TBST, at room temperature [ 40 ].…”

Ex Situ Conservation and Genetic Rescue of Endangered Polish Cattle and Pig Breeds with the Aid of Modern Reproductive Biotechnology – A Review

“…To sum up, the efficiency of intra-species and inter-species somatic cell cloning of domesticated mammals is affected, to a great extent, by three factors: the provenance of frozen/thawed and ex vivo-expanded nuclear donor cell lines (NDCLs) that provide genetic resources from different livestock species or breeds (Samiec and Skrzyszowska, 2010;Li et al, 2013;Olivera et al, 2016;Lee et al, 2019), the morphological, cytogenetic, cytophysiological and molecular quality parameters of the NDCLs (Samiec et al, 2013(Samiec et al, , 2019Zhang et al, 2019;Wiater et al, 2021); and the subsequent incidence of programmed (apoptotic or autophagic) cell death in the development of cloned embryos (Chi et al, 2017;Samiec et al 2015;Jeong et al, 2020). This efficiency is also dependent on such molecular mechanisms as the epigenetic ability of the nuclear donor cell-inherited genome to be reprogrammed in SCNT-derived oocytes and result in cloned embryos (Samiec and Skrzyszowska, 2018;Deng et al, 2020;Sampaio et al, 2020;Wang et al, 2020b) and the intergenomic crosstalk between nuclear and mitochondrial DNA fractions in the host cytoplasm of nuclear recipient oocytes and the blastomeres of the developing cloned embryos (Srirattana et al, 2011;Samiec and Skrzyszowska, 2021;Takeda, 2019;Xu et al, 2019;Magalhães et al, 2020) (Figure 1).…”

Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine

“…This calls for studies aimed at the precise determination of the conditions that facilitate epigenetic reprogramming in the nuclear donor cell genome during the pre- and postimplantation development of SCNT-generated embryos and fetuses of different mammalian species, including the domestic goat [ 5 , 6 , 7 , 8 , 9 , 10 ]. Promising results were achieved by investigations that focused on the use of extrinsic nonselective agents for stimulating the epigenetically regulated transcriptional activity of genomic DNA in both nuclear donor somatic cells and SCNT-cloned embryos [ 11 , 12 , 13 , 14 , 15 ].…”