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

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human alpha 1,2-fucosyltransferase (rh alpha 1,2-FT) and alpha-galactosidase A (rh alpha-Gal A) immune system enzymes but also Gal alpha 1 -> 3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2xhGLA 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 rh alpha 1,2-FT and rh alpha-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Gal alpha 1 -> 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 hFUT2xhGLA and nTG samples, the expression levels of alpha 1,2-FT and alpha-Gal A proteins in TSA(+) cells were more than twofold higher in comparison to TSA(-) cells. Moreover, a much lower expression of the Gal alpha 1 -> 3Gal epitopes was shown in TSA(-) hFUT2xhGLA cells as compared to the TSA(-) nTG group. Interestingly, the levels of Gal alpha 1 -> 3Gal expression in TSA-treated hFUT2xhGLA 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.