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

Abstract

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

Folate receptor‐1 is vital for developmental competence of goat embryos

“…Despite effective knockdown, 5.87% of embryos was able to develop into blastocysts, possibly due to the presence of maternally contributed FOLR1 which may eventually exhausted until blastocyst stage. Our results are in line with a previous study (Strandgaard et al, 2017), which demonstrated the compromised ability of mouse embryos to develop to blastocyst stage after TA B L E 1 Effect of FOLR1 knockdown on the rate of blastocyst production injection (Kharche et al, 2016;Magata et al, 2019;Menéndez-Blanco et al, 2020;Shirazi et al, 2018) and cloning by somatic cell nuclear transfer (Gorczyca et al, 2021;Hernández Martínez et al, 2020;Samiec et al, 2019;Skrzyszowska & Samiec, 2020Wiater et al, 2021).…”

“…The future studies at our laboratory may decipher some of these questions in order to thoroughly elucidate the importance of FOLR1 as a pivotal biomarker suitable for prediction of augmented susceptibility of embryonic genomes to efficient epigenetic reprogramming of their transcriptional activities. The latter could enable the embryologists to estimate the increased developmental competences and molecular quality of caprine and other mammalian species embryos produced by such modern assisted reproductive technologies as microsurgical IVF by intracytoplasmic sperm injection (Kharche et al., 2016; Magata et al., 2019; Menéndez‐Blanco et al., 2020; Shirazi et al., 2018) and cloning by somatic cell nuclear transfer (Gorczyca et al., 2021; Hernández Martínez et al., 2020; Samiec et al., 2019; Skrzyszowska & Samiec, 2020, 2021; Wiater et al., 2021).…”

Effects of IGF-1 on the Three-Dimensional Culture of Ovarian Preantral Follicles and Superovulation Rates in Mice

“…The results of the current research have proven that designing modern and powerful approaches to the murine ex vivo 3D model of IGF-1-assisted growth and maturation of ovarian secondary preantral follicles in order to obtain satisfactory outcomes of IVM might be reliable and feasible for the purposes of achieving high efficiency rates in such novel ARTs as IVF/ICSI in humans and other mammalian species [ 65 , 66 , 67 , 68 ] or somatic cell cloning in other mammalian species [ 69 , 70 , 71 , 72 ].…”

Oocyte Penetration Speed Optimization Based on Intracellular Strain

“…Collectively, the results of the current investigation aimed to devise and optimize intracellular strain-related parameters that turned out to vary depending on different speeds of penetrating the porcine oocytes with the use of robotic (i.e., motorized) micro-manipulators might contribute to improvement of the efficiency of a variety of modern assisted reproductive technologies (ARTs). Taking the above-mentioned findings into consideration, the augmented efficiencies of microsurgical in vitro fertilization by ICSI and cloning by somatic cell nuclear transfer (SCNT) will be of especially great importance not only in pigs but also in other mammalian species [25][26][27][28][29][30][31].…”