# **Molecular Mechanism and Application of Somatic Cell Cloning in Mammals**

Edited by

Marcin Samiec and Maria Skrzyszowska

Printed Edition of the Special Issue Published in *International Journal of Molecular Sciences*

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## **Molecular Mechanism and Application of Somatic Cell Cloning in Mammals**

## **Molecular Mechanism and Application of Somatic Cell Cloning in Mammals**

Editors

**Marcin Samiec Maria Skrzyszowska**

MDPI ' Basel ' Beijing ' Wuhan ' Barcelona ' Belgrade ' Manchester ' Tokyo ' Cluj ' Tianjin

*Editors* Marcin Samiec Department of Reproductive Biotechnology and Cryoconservation National Research Institute of Animal Production Balice near Krakow´ Poland

Maria Skrzyszowska Department of Reproductive Biotechnology and Cryoconservation National Research Institute of Animal Production Balice near Krakow´ Poland

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: www.mdpi.com/journal/ ijms/special issues/Somatic Cell Cloning).

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## **Contents**

The Molecular Quality and Mitochondrial Activity of Porcine Cumulus–Oocyte Complexes Are Affected by Their Exposure to Three Endocrine-Active Compounds under 3D In Vitro Maturation Conditions

Reprinted from: *Int. J. Mol. Sci.* **2022**, *23*, 4572, doi:10.3390/ijms23094572 . . . . . . . . . . . . . . **141**

## **About the Editors**

#### **Marcin Samiec**

Prof. Dr. Marcin Samiec is a biotechnologist and embryologist with long-term and broad experience. He is employed as a Senior Scientist (Professor) at the Department of Reproductive Biotechnology and Cryoconservation of the National Research Institute of Animal Production in Balice near Krakow, Poland. His areas of expertise and interest include: (1) reproductive biology ´ and biotechnology in different mammalian species (especially pigs, goats, cattle, rabbits and horses); (2) assisted reproductive technologies (ARTs) (experimental and applied embryology); (3) intra- and interspecies somatic cell cloning by somatic cell nuclear transfer (SCNT); (4) transgenic research; (5) parthenogenetic activation of oocytes (methods and molecular mechanisms of oocyte activation); (6) in vitro embryo production (IVP): in vitro oocyte maturation (IVM), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and in vitro embryo culture (IVC); (7) stem cell research; (8) epigenetic and molecular aspects of embryonic development: epigenetics in developmental biology, epigenetic modulation of nuclear donor cells (somatic cells, stem cells), SCNT-derived oocytes or cloned embryos; (9) unravelling the transcriptomic and proteomic profiles in nuclear donor cell lines (somatic and stem cell lines) and nuclear recipient oocytes; (10) the ART-mediated programs of ex situ biodiversity conservation to protect and maintain the genetic resources of wild-living and domesticated mammalian species. He is the author and ad hoc reviewer of many research articles published in high-impact journals indexed in the JCR, WoS and Scopus databases. He is also the author of several patents granted for biotechnological/embryological inventions (indexed in the WoS database).

#### **Maria Skrzyszowska**

Prof. Dr. Maria Skrzyszowska is an embryologist with many years of experience. She is employed as a Senior Scientist (Professor) at Department of Reproductive Biotechnology and Cryoconservation of National Research Institute of Animal Production in Balice near Krakow, Poland. ´ She is a specialist in the fields of: (1) animal reproduction biotechnology; (2) somatic cell cloning in different mammalian species; (3) embryonic cell cloning in different mammalian species, including embryo bisection methods; (4) in vitro embryo production; (5) in vitro oocyte maturation and ICSI-mediated in vitro fertilization; (6) transgenesis; (7) modern strategies to assess the molecular quality of somatic cell lines and female gametes for the purposes of advanced ARTs. She is the author or co-author of many research articles published in high-impact journals indexed in JCR, WoS and Scopus databases and several patents for embryological inventions (indexed in WoS database).

## **Preface to "Molecular Mechanism and Application of Somatic Cell Cloning in Mammals"**

The paramount goal of the current book (in the form of a scientific monograph) is to comprehensively provide the state of the art and research highlights of the molecular factors and their biological networks determining the effectiveness of SCNT-mediated cloning from the perspective of mechanistic insights into genomic, epigenomic, transcriptomic and proteomic landscapes specific for nuclear donor cells, nuclear recipient oocytes and nuclear-transferred embryos. Widely unravelling the multi-faceted mechanisms underlying inter-genomic, inter-epigenomic, inter-transcriptomic and inter-proteomic crosstalk between the above-indicated factors might be a pivotal stimulus triggering the augmentation of in vitro and in vivo molecular capabilities of donor cell nuclei to be epigenetically reprogrammed in SCNT-derived oocytes, embryos, fetuses and offspring. In turn, this would enable scientists to increase the applicability of SCNT-based assisted reproductive technologies (ARTs) to a broad spectrum of research areas and interdisciplinary fields such as: (1) experimental and applied embryology; (2) biotechnology; (3) transgenics; (4) biomedicine; (5) biopharmacy; and (6) practical activities designed to develop and optimize the ex vivo and in vivo models focused on recognizing the etiopathogenesis, phenotypic and genotypic backgrounds of human and other mammalian hereditary or acquired diseases.

### **Marcin Samiec and Maria Skrzyszowska**

*Editors*

### *Editorial* **Molecular Mechanism and Application of Somatic Cell Cloning in Mammals—Past, Present and Future**

**Marcin Samiec**

Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Krakowska 1 Street, 32-083 Balice, Poland<EMAIL_ADDRESS>
Thus far, nearly 25 mammalian species have been cloned by intra- or interspecies somatic cell nuclear transfer (SCNT). Among them, non-transgenic and transgenic representatives of such domesticated and wild-living animals that have been propagated and/or multiplied by intraspecific or interspecific SCNT-based cloning are:

Despite the above-indicated abundant variety of SCNT-derived mammalian species, the effectiveness of SCNT-based cloning remains immensely or considerably low and oscillates between 0.1% and 5%, while estimating the outcomes of offspring born in relation to the total numbers of nuclear-transferred oocytes [30,31]. For this reason, at the present stage of investigations, extensive efforts are being undertaken to achieve considerable scientific breakthroughs, which would enable researchers to not only tremendously increase the ex vivo and in vivo developmental competences, but also to remarkably ameliorate the parameters related to the cytological, molecular and epigenetic qualities of SCNT-generated mammalian embryos. Only such a crucial turning point or a substantial research game changer would open up new possibilities for both improving the overall efficiency of SCNTbased cloning and, as a consequence, play an increasingly important role as an assisted reproductive technology (ART) which is characterized by a broad spectrum of applicability in embryology, biotechnology, transgenics and biomedicine [32,33].

**Citation:** Samiec, M. Molecular Mechanism and Application of Somatic Cell Cloning in Mammals—Past, Present and Future. *Int. J. Mol. Sci.* **2022**, *23*, 13786. https://doi.org/10.3390/ ijms232213786

Received: 2 November 2022 Accepted: 7 November 2022 Published: 9 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

It is beyond any doubt that the relatively or extremely low efficiency of mammalian SCNT-mediated cloning, including both its intra- and interspecies model, can only be improved by comprehensively recognizing molecular and epigenetic determinants and mechanisms affecting the developmental competences of SCNT-derived embryos [34]. A wide range of biological and molecular factors predestine and predominantly bias the biotechnological suitability of nuclear donor cells and nuclear recipient oocytes for SCNTmediated ARTs. The extent of this suitability is measured and directly depends on the developmental capacity and quality parameters pinpointed for nuclear-transferred oocytes and corresponding somatic-cell-cloned embryos in different mammalian species [35]. The main impact on the development of cloned embryos is exerted by the type and provenance of nuclear donor cells [36–38]. In this context, an important role is played by the strategies used to artificially synchronize the mitotic cycle of nuclear donor cells expanded ex vivo at the G0/G1 stages [39,40]. Notably, the developmental outcomes of somatic-cell-cloned embryos are largely determined by the molecular quality parameters reflected in the incidence of apoptotic cell death and oxidative stress processes in the nuclear donor cells and SCNT-derived embryos cultured in vitro [40–43]. Additionally, it is worth highlighting that the developmental capability of cloned embryos is remarkably biased by the molecular quality of metaphase-II stage nuclear recipient oocytes, which largely depends on coordination between the processes of meiotic, cytoplasmic and epigenomic maturation [44–46]. Not without significance is the tremendous influence of the approaches applied to artificially activate the embryo-specific developmental program of SCNT-derived oocytes on the efficacy of propagating cloned embryos and their molecular quality [47–49]. Furthermore, the effectiveness of generating somatic-cell-cloned embryos results from the capabilities of donor cell nuclei to epigenetically reprogram their transcriptomic signatures in the cytoplasm of SCNT-derived oocytes and the blastomeres of corresponding cloned embryos [50,51]. In turn, the epigenomic reprogrammability of transcriptional activity within donor cell nuclei has been proven to be strongly affected by the molecular network of interrelations between nuclear and mitochondrial genomes that has been established during the early embryonic development of activated SCNT-derived oocytes [52–54]. Finally, as a consequence of applying a wide variety of methods focused on modulating/transforming the transcriptional activities of donor cell nuclear genomes by extrinsic epigenetic modifiers such as non-selective inhibitors of histone deacetylases (HDACi) and/or non-selective inhibitors of DNA methyltransferases (DNMTi), the enhanced capabilities of donor cell nuclei to correctly and faithfully reprogram their transcriptomic profiles in SCNT-derived embryos have been shown [31,55,56].

In summary, this Special Issue will publish research articles and comprehensive review papers aimed at highlighting the state of the art and mechanistic insights into precisely identifying and unravelling a wide array of genomic, epigenomic, transcriptomic and proteomic factors which cumulatively determine the molecular parameters which are of paramount importance for the quality of nuclear donor cells, nuclear recipient oocytes and SCNT-derived embryos. Thoroughly deciphering the multifaceted nature of all the aforementioned factors and insightful interpretation of the biological crosstalk between them can finally bias the augmentation of the overall efficiency of SCNT-based cloning. This is a preponderant condition indispensable for the practical implementation of SCNT-mediated ARTs to various research fields and interdisciplinary studies at the interface of experimental and applied embryology, biotechnology, transgenics, biomedicine, biopharmacology, the creation of animal models for etiopathogenesis and physiopathology of human diseases and the genetic rescue and/or resurrection of endangered/extinct mammalian species.

**Funding:** The present study was financially supported by research grant No. 04-19-11-21 from the National Research Institute of Animal Production in Balice near Kraków, Poland, to M.S. (Marcin Samiec). **Conflicts of Interest:** The author declares no conflict of interest. The author had no financial or other relationship with other people or organizations that might inappropriately influence this work. The funders had no role in the writing of the manuscript or in the decision to publish the results.

#### **References**

## *Review* **Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals**

**Marcin Samiec \* and Maria Skrzyszowska**

Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, 32-083 Kraków, Poland<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

**Keywords:** cloned mammalian embryo; SCNT-derived progeny; mtDNA; nuclear–mitochondrial interaction; epigenetic reprogrammability; telomere shortening/attrition

#### **1. Biotechnological Possibilities of Applying the Techniques of Somatic Cell Nuclear Transfer (SCNT) to Produce Cloned Mammalian Species**

The somatic cell cloning technique is a method of embryonic genome engineering. Unlike animal transgenesis, it involves micromanipulation not of individual nuclear DNA genes but of the whole nuclear and/or mitochondrial genome of both interphase nuclear donor somatic cells and female germ cells (in vitro- or in vivo-matured oocytes arrested at metaphase II), which are used as recipients of exogenous genetic material. Of all mammalian cloning techniques, somatic cell cloning can result in producing the largest numbers of genetically identical individuals that are designated as clones. In the somatic cell cloning of mammals, nuclear donor cells are available in practically unlimited quantities. Tissue samples obtained by biopsy from adult animals or fetuses are composed of hundreds of thousands cells, which can be further multiplied/expanded in vitro. Furthermore, when

**Citation:** Samiec, M.; Skrzyszowska, M. Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals. *Int. J. Mol. Sci.* **2021**, *22*, 3099. https://doi.org/ 10.3390/ijms22063099

Academic Editor: Sato Masahiro

Received: 4 March 2021 Accepted: 16 March 2021 Published: 18 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cloning certain adult animals, tissue may be biopsied repeatedly to produce identical clones every time [1–7].

Animal cloning by somatic cell nuclear transfer (SCNT), which avoids the sexual reproduction pathway, offers the opportunity to obtain monogenetic offspring derived not only from adult animals of high genetic merit but also from genetically transformed (transgenic) specimens. Over the last 24 years, intra- and interspecies cloning via SCNT resulted in a fairly large number of transgenic and non-transgenic offspring, not only in various species or infertile interspecific hybrids (bastards) of domesticated animals, such as

Explanation of the mechanisms underlying intergenomic communication between nuclear and mitochondrial DNA molecules in cloned embryos and recognition/identification of the determinants affecting aberrant epigenetic reprogrammability of chromosomal telomeres will be suitable and reliable for resolving or reducing the imperfections in the generation of cloned embryos, conceptuses, and offspring by using SCNT technology. Moreover, the development of efficient strategies applied to the cryopreservation of nuclear donor somatic cells, nuclear-transferred oocytes reconstructed with somatic cells, and somatic cell-cloned embryos seems to be an inevitable progressive step contributing to the expedition of future large-scale attempts aimed to more successfully produce and multiply mammalian cloned offspring. The latter seems to be a sine qua non condition that allows one to more efficiently use SCNT-based assisted reproductive technology not only for transgenic, biotechnological, biomedical, and biopharmaceutical research but also for the ex situ conservation of biodiversity in both anthropogenic (agricultural) and non-anthropogenic (wild) ecosystems.

#### **2. Dependence of Epigenetic Mechanisms Underlying Somatic Cell Nuclear Reprogramming and Intergenomic Communication between Nuclear and Mitochondrial DNA Fractions in Cloned Embryos on Various Approaches to Reconstruction of Enucleated Oocytes**

In the reconstruction of enucleated oocytes (cytoplasts/ooplasts) by SCNT, the original genetic material is replaced with the somatic cell-inherited nuclear genome. Different approaches to SCNT are used to generate nuclear-transferred oocytes, i.e., oocytes reconstructed with somatic cell nuclei and the resultant cloned embryos (Table 1). The most common procedure is a relatively low-invasive method of SCNT based on the fusion of cytoplast–nuclear donor cell couplets that is induced by electric pulses [34,97–101] (Table 1). An alternative reconstruction method is a much more invasive microsurgical procedure, in which whole nuclear donor cells [102,103] or somatic-cell-derived karyoplasts [29,44,82,104,105] are microinjected directly into the cytoplasm of enucleated oocytes (Table 1). The karyoplast is a live membrane-bound structure formed as a result of mechanically induced lysis of the whole somatic cell. It contains the interphase cell nucleus or metaphase chromosomes that are surrounded only by a thin layer of the perinuclear cytoplasm (the so-called perikaryon) [27,105–108].

Whatever the method used, the reconstruction of ooplasts results in the combination and mingling (hybridization) of cytoplasmic environments of the ooplast and intact somatic cell or karyoplast isolated from the whole nuclear donor cell. As a result, a nuclear– cytoplasmic/nuclear–ooplasmic hybrid (i.e., cloned cybrid) is formed. This hybrid cell, formed by the hybridization of cytoplasmic microenvironments of the cells derived from two different developmental lines: gametogenic (germinal) and somatogenic (somatic), is referred to as a reconstructed or reconstituted oocyte or cybrid cloned zygote. As the mitotic cycle of nuclear donor somatic cells (artificially arrested at the G0 phase) is characterized by "latent" transcriptional activity, inhibited proliferative growth, and a slower metabolism of all organelles, the meiotic cycle of nuclear recipient oocytes also undergoes transient and reversible arresting at the metaphase II (MII) stage. At this stage of meiosis, the processes of advanced transcriptional suppression of genomic DNA take place as a result of attaining nuclear and ooplasmic maturity states. Proper coordination of the cytophysiological state of somatic cells or the karyoplasts isolated from them, and of the cytophysiological state of ooplasts during the reconstruction of cloned cybrids, results from the hybridization of the cytoplasmic environments of nuclear donor cells at the G0 phase of mitosis and of enucleated nuclear recipient oocytes at the MII stage of meiosis [27,98,101,109,110].

Techniques of enucleated oocyte reconstruction may largely affect molecular mechanisms of nuclear chromatin rearrangement, which include both its structural remodeling and epigenetic reprogramming of genomic DNA [99,102,104,107,111–113]. Hybridizing the cytoplasmic environment of two cells at different stages of the division cycle interferes with the cell cycle controlling mechanisms and carries the risk of abnormalities further into the development of the cybrid cloned zygote. However, not only does the proper selection of the cytophysiological states of somatic cells/karyoplasts and ooplasts during the reconstruction of cloned cybrids reduce genomic instability, rendering the genome less vulnerable to mutations, but it also reduces the degree of asynchrony in nuclear– cytoplasmic interactions and decreases the frequency of abnormal epigenome-dependent rearrangements of exogenous nuclear chromatin [114–120].

In contrast to electrofusion, intraooplasmic microinjection of karyoplasts allows for the selective removal of a large part of the cytoplasm of nuclear donor cells, thus enabling relative thinning of the remnants of the somatic cell cytoplasm in a cytosolic microenvironment of the ooplast and early zygote. The direct consequence of this is that the adverse effect of cytoplasmic components of the somatic cell on remodeling and reprogramming of the transferred somatic cell nucleus, and thereby on the development of the reconstituted embryo, is avoided. Where nuclei of relatively small-diameter somatic cells are transplanted (e.g., cumulus oophorus cells, mural granulosa cells, and serum-starved fibroblast cells), the method of choice is the intraooplasmic microinjection of karyoplasts or whole nuclear donor cells [80,98,102–104,112,121,122]. Taking into account the above-mentioned finding, the in vitro developmental potential of cloned pig embryos that had been reconstructed by direct intraooplasmic microinjection of somatic cell-descended karyoplasts or whole tiny somatic cells was shown to be relatively higher in relation to cloned embryos produced by the electrofusion of somatic cell–ooplast couplets [29,102,107]. The small diameter of the above types of somatic cells is the reason for a considerably reduced contact surface area with the plasmalemma of enucleated oocytes (oolemma), which reduces the percentage of fused ooplast–nuclear donor cell complexes. In turn, the direct microinjection of karyoplasts or whole small-diameter somatic cells into the cytoplasm of enucleated oocytes avoids technical problems (resulting from inadequate adhesion of plasma membranes), which have the greatest limiting effect on the efficiency of electrofusion of nuclear donor cells with cytoplasts [102–104,122].

The direct microinjection of somatic cell nuclei into the cytoplasm of enucleated oocytes has the added advantage of being the "cleanest" of all nuclear transplantation methods. It requires no physicochemical transducers, which often have adverse effects by reducing the in vitro developmental potential of mammalian cloned embryos. For the cell electrofusion technique, all components of the donor cell (both nuclear and cytoplasmic components: organelles and cytoskeletal elements) become an integral part of the oocyte. In contrast, for intraooplasmic microinjection of karyoplasts, plasmalemma and the vast majority of the cytoplasmic material of the nuclear donor cell is rejected following cell lysis. Therefore, only trace amounts of residual cytoplasm, in the form of a narrow rim of membrane-bound protoplasm around the cell nucleus, are introduced as a small karyoplast into the enucleated oocyte. This is of prime importance in some studies that examine nuclear–cytoplasmic interactions in mammalian cloned cybrids [29,44,81,82,104,107,111,122].

The basic paradigm underlying the somatic cell cloning of mammals is the scientific thesis that the donor cell nucleus has to be completely reprogrammed epigenetically by specific factors of the oocyte's origin in order to support the development of the cybrid cloned zygote to term. A considerable portion of the protein nucleoplasmic (karyolymphatic) factors and cytosolic factors of the somatic cell, which are engaged directly or indirectly in the mechanisms underlying epigenetic reprogramming of donor cell genome, is associated with nuclear chromatin. The qualitative and quantitative composition of these factors within the somatic cell changes together with progressing cytodifferentiation. When the whole donor cell is fused with the enucleated oocyte, those specific factors of somatic cell are also transferred into the cytoplasm of the nuclear recipient oocyte. As a result of this, they may block the endogenous oocyte factors from supporting proper remodeling and reprogramming the epigenetic profile, which is characteristic of a foreign nucleus of a terminally differentiated somatic cell, toward an epigenetic status typical of the nucleus of totipotent stem cells such as the zygote [28,102,123–127]. Exogenous nucleoplasmic and cytoplasmic factors derived from the nuclear donor cell, which are responsible for modulating the epigenetic status of genomic DNA, are incorporated together with oocyte mRNA transcripts and proteins, into the remodeled nucleus of the somatic cell (the so-called pseudo-pronucleus). The pseudo-pronucleus is formed following artificial activation of the embryonic developmental program of the reconstructed oocyte [1,128–137]. In turn, an overabundance of the somatic cell-derived agents modulating the epigenetic

profile of the donor nucleus may remarkably reduce the concentration and activity of the oocyte's epigenetic factors. Thus, it may diminish the incidence of complete epigenetic reprogramming of transcriptional activity of the somatic cell nucleus in the developing cloned embryo [112,132–141].

#### **3. Inheritance of the Mitochondrial Genome and Intergenomic Communication between Mitochondrial and Nuclear DNA Fractions during the Development of Cloned Embryos**

The increased competence of the oocyte cytoplasm for epigenetic remodeling and reprogramming the somatic cell-inherited nuclear and mitochondrial genomes in cybrid cloned zygotes is a sine qua non condition for correctly inducing the developmental program specific for mammalian SCNT embryos [142–154].

Mitochondria are semiautonomous organelles that contain their own genetic material in the form of double-stranded (α-helix) circular DNA molecules (mtDNAs) of about 16,300–16,500 base pairs (bp). The mitochondrial DNA encodes 13 proteins, 22 tRNAs, and 2 rRNAs. Up to 95% of proteins, which are the products of the cytoplasmic translation system encoded by nuclear DNA, are involved in biogenesis and cytophysiological functions of mitochondria [155–157]. The copy number of mitochondrial genome in a typical mammalian somatic cell is approximately 2–5 <sup>×</sup> <sup>10</sup><sup>3</sup> , whereas the number of mtDNA molecules in a meiotically matured (MII-stage) oocyte is about 1.6 <sup>×</sup> <sup>10</sup><sup>5</sup> in mice, 2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> in cattle, 3–5 <sup>×</sup> <sup>10</sup><sup>5</sup> in pigs, and 3–8 <sup>×</sup> <sup>10</sup><sup>5</sup> in humans. The number of mitochondria in the somatic cell averages 1 <sup>×</sup> <sup>10</sup><sup>3</sup> , and one organelle harbors between 1 and 10 mtDNA molecules. In turn, a single mitochondrion in the meiotically matured oocyte contains from one to two copies of the mitochondrial genome, which confirms that the abundance of the intraooplasmic population of these organelles is generally equivalent to the total pool of mtDNA molecules of an unfertilized mammalian oocyte [106,139,158,159].

In the procedure of cloning by SCNT, mitochondria of nuclear donor cells are transplanted with the nuclear genetic apparatus into the cytoplasm of enucleated recipient oocytes. Irrespective of the method used for the reconstruction of enucleated oocytes (Table 1), this step of the SCNT procedure always results in the conjunction and mingling (hybridization) of cytoplasmic environments of the ooplast and somatic cell or karyoplast. After its intraooplasmic microinjection, the karyoplast may also be a source of mitochondria (mitochondrial genome) of heteroplasmic origin. Therefore, a reconstructed cloned embryo, which from a cytological viewpoint is a cytoplasmic hybrid (cybrid), harbors the mitochondrial genome of both maternal (oocyte's) and exogenous origin (i.e., introduced together with the nuclear donor cell) [107,111,160–162]. In cloned embryos, fetuses, and offspring, mitochondria are primarily inherited with ooplasmic material. In turn, probably during the first few mitotic cleavage divisions, mitochondria derived from nuclear donor cells are rapidly eliminated from the cytoplasm of embryonic cells at the anaphase stage. The removal of somatic cell-inherited mitochondria largely depends on the polyubiquitination of specific protein substrates. For that reason, the presence of the somatogenic mitochondrial genome in the cells of cloned blastocysts is difficult to detect by genetic engineering techniques [133,158,163,164]. As a consequence, the uniparental inheritance of extranuclear genetic information in dividing cybrid cloned zygotes is regulated by the biodegradation of ubiquitin-labeled mitochondrial proteins (including ribonucleoproteins) and the nucleolysis of mtDNA molecules that are deprived of histones and non-histone proteins. The proteolytic degradation of mitochondria of heteroplasmic (allogeneic) origin is catalyzed by a complex proteasomal system in each blastomere of cloned embryos. This system is characterized by a Svedberg sedimentation coefficient of 26 and designated as a 26S proteasome. The mechanism of nucleolytic biodestruction of all the somatic-cellderived mtDNA copies is determined by normal function of the intracellular lysosomal cycle, which is related to the exocytosis of endosomal vesicles. The ultimate outcome of this reaction is the removal from embryonic cells of the exogenous mtDNA fractions, which had previously been subjected to internucleosomal fragmentation into short oligonucleotide segments. The preimplantation-stage selective segregation of the mitochondrial genome

stemming from nuclear donor cells that is indirectly induced by the anaphase-promoting complex/cyclosome (APC/C) gradually leads to the establishment of cellular mtDNA homoplasmy in cloned embryos reconstituted with somatic cell nuclei. It is noteworthy that APC/C undergoes the heterodimerization with cyclin-dependent kinase cdc20 and is an integral part of the polysubunit enzymatic complex of ubiquitin ligase. Only occasionally could the lasting hybridization of allogeneic mtDNA copies (the so-called mtDNA heteroplasmy) be identified in the pre- and postnatal period of ontogenetic development of mammalian cloned specimens. This phenomenon of intracellular mtDNA heteroplasmy resulted from synergism/complementarity in the intergenomic communication between mtDNA molecules inherited with both nuclear donor cell cytoplasm and nuclear recipient cell ooplasm [155,165–168].

There are several species-specific epigenetic factors present in the oocyte cytoplasm that may contribute to nuclear–cytoplasmic incompatibilities either immediately after somatic cell nuclear transfer or at later stages of cloned embryo development [105,136,169,170]. In turn, this potential lack of coordination in the interactions of nuclear and cytosolic factors of cybrid cloned zygotes is probably one of the reasons for the limited practical application of the somatic cell cloning technique. It has been demonstrated that maternally inherited mtDNA molecules accumulated in the mitochondrial reservoirs of the oocyte cytosol play an important role in nuclear–ooplasmic asynchrony. This asynchrony involves incompatibilities in both the epigenetic modifications of the somatic genome supporting the developmental program of reconstituted cybrids and a lack of synergy in the molecular mechanisms controlling the karyokinesis and cytokinesis restriction points. These restriction points related to the anaphase segregation of somatic cell-derived chromosomes and asymmetrical telophase division of the cloned cybrid (nuclear–ooplasmic hybrid) that encompasses the expulsion of the pseudo-polar body into perivitelline space are collectively responsible for coordinated pseudomeiotic to mitotic cycle transition following activation of the reconstituted oocyte [134,156].

Moreover, the presence of an oocyte-derived mitochondrial genetic apparatus has been shown to influence the implantation of cloned embryos in the endometrium of a recipient female's uteri. For that reason, the deleterious effect, on the preimplantation development of cloned embryos, of heterogeneous mtDNA sources as a result of possible mitochondrial heteroplasmy in the reconstructed nuclear–cytoplasmic hybrids should not be discounted [139,160,161,169]. That is why the production of nuclear-transferred embryos, fetuses, and offspring with a precisely defined profile of nucleotide sequences in regulatory or coding segments of the nuclear and/or mitochondrial genome seems to be valuable tool. This tool can be suitable for experimentally dissecting the effects of not only nuclear and cytoplasmic genetic/epigenetic components but also the intrauterine environment of recipient females on embryonic, fetal, and postnatal development of cloned specimens [2,155,158,171].

Therefore, in the hybrid cytoplasmic environment of cloned zygotes, genetically different fractions of mitochondrial DNA of maternal (oocyte's) origin were found to coexist with those derived from the cytoplasm of allogeneic somatic cells. Although this extranuclear (mitochondrial) genetic apparatus of cloned nuclear–ooplasmic hybrids contains small (approximately 0.01%) amounts of a cell's genetic information, this mtDNA-dependent genetic information is completely different from information recorded in the nucleotide sequences of nuclear DNA. The latter provides approximately 99.99% of the cellular genome. In this respect, nuclear transplantation of allogeneic somatic cells into enucleated recipient oocytes (where nuclear donor cells and oocytes are derived from genetically different animals of the same species) gives rise to generating nuclear–cytoplasmic hybrids, which are characterized by heterogeneous mtDNA copies. In view of the fact that such heteroplasmic cloned cybrids develop into embryos with cellular mtDNA heteroplasmy, this may lead to apparent genotypic and phenotypic identity/compatibility of the cloned offspring (only in terms of traits determined by nuclear genome-dependent inheritance). Such cloned offspring exhibits a degree of variation/incompatibility with regard to phenotypic traits

determined by cytoplasmic (extranuclear) inheritance. The latter is dependent on the mitochondrial genotype known as the mitotype [105,157,159,160,167].

Different possible patterns/scenarios of extranuclear (cytoplasmic) inheritance of mtDNA fractions have been presented (see Figure 1 (for intraspecies cloning by SCNT) [2,158,165,172], Figure 2 (for interspecies SCNT using nuclear donor cells and recipient oocytes derived from closely related mammalian species) [163,170,171,173,174], and Figure 3 (for interspecies SCNT using nuclear donor cells and recipient oocytes derived from phylogenetically distant mammalian species) [162,164,175,176]).

The "ideal" clone can be generated only in a situation where the nuclei of its own (autogeneic) somatic cells are transferred into enucleated recipient oocytes. Put another way, such a cloned specimen can be produced when nuclear donor cells and oocytes originate from genetically identical individuals of a mammalian species, i.e., from monosexual (female) individuals. It is necessary to stress that completely homoplasmic cybrid cloned zygotes can only be created from the oocytes reconstructed in such a manner. The latter are characterized by homogeneous fractions of mtDNA molecules. The artificial activation of such nuclear–cytoplasmic hybrids results in the development of cloned embryos displaying cellular mtDNA homoplasmy. This naturally results in complete genotypic and phenotypic identity/compatibility of somatic cell-cloned fetuses and the resultant offspring. Taking into consideration the previously mentioned findings, only in the case of mammalian cloned females does the mitotype exhibit a homogeneous pattern of coding and regulatory sequences in all mtDNA copies of the somatic and germ cell lines. This condition can only be met assuming that during ontogenesis, the mitochondrial genome will not undergo spontaneous point mutations or those induced by reactive oxygen species [2,42,106,133,166,168,172,177].

Among the reasons for genetic diversification between the cloned specimens generated (somatic clones) and individuals subjected to somatic cell cloning (i.e., donors of somatic cells for SCNT procedure), mention should be made of the effect of mitochondrial (extranuclear/extrachromosomal) inheritance and the impact of intrauterine environment of recipient females receiving cloned embryos. Extranuclear inheritance of genetic material results from the microsurgical, random introduction of foreign mtDNA copies with the nuclear donor cell cytoplasm into the cytoplasmic environment of recipient oocyte. The mismatch of the mitochondrial genome molecules of maternal (oocyte's) origin and of somatogenic (nuclear donor cell) origin, i.e., mtDNA heteroplasmy, leads to inter-specimen diversification within the mitotype. This results in intra-population and inter-population genetic and phenotypic variability dependent on the mitochondrial genome [134,155–157,161]. The phenotypic differences between somatic clones and specimens undergoing SCNT are also contributed by different morphological, anatomotopographical, histological, physiological, endocrinological, embryotrophic, and immunological considerations associated with the reproductive system of recipient surrogates. Moreover, transplacental leakage of leukocyte and erythroblast mitochondria from the blood stream of recipient surrogates to the blood stream of cloned fetuses is often observed. This type of leukocyte–erythroblast chimerism results both from mtDNA heteroplasmy in peripheral blood cells and from genetic mosaicism within subpopulations of nucleated hematopoietic cells (i.e., hematopoietic karyocytes). Such chimerism may also have a certain effect on differences in the mitotype of cloned progeny [139,160,165,169].

**Figure 1.** Intraspecies somatic cell cloning, in which the inheritance of allogeneic (homologous) mtDNAs stemming from genetically different nuclear recipient oocytes and nuclear donor cells is still incompletely recognized during preimplantation development of nuclear-transferred pig embryos. In the vast majority of intraspecies (porcine) cloned embryos, mitochondrial genome primarily arises from the nuclear recipient oocytes, whereas in their other counterparts, mtDNA copies appear to be inherited heteroplasmically (i.e., both from nuclear donor cells and from recipient ooplasm). *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 12 of 29

**Figure 2.** Interspecies (intrafamily and intragenus) somatic cell cloning, in which nuclear donor cells and recipient oocytes are recovered from phylogenetically consanguineous species (i.e., wild boar and domestic pig, respectively). Porcine oocyte-derived mitochondrial DNA (mtDNA) is inherited predominantly during preimplantation development of interspecies (wild boar→pig) cloned embryos, leading to intracellular mtDNA homoplasmy at the blastocyst stage. **Figure 2.** Interspecies (intrafamily and intragenus) somatic cell cloning, in which nuclear donor cells and recipient oocytes are recovered from phylogenetically consanguineous species (i.e., wild boar and domestic pig, respectively). Porcine oocyte-derived mitochondrial DNA (mtDNA) is inherited predominantly during preimplantation development of interspecies (wild boar→pig) cloned embryos, leading to intracellular mtDNA homoplasmy at the blastocyst stage. *Int. J. Mol. Sci.* **2021**, *22*, 3099 *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 13 of 29

**Figure 3.** Interspecies (interfamily and intergenus) somatic cell cloning, in which nuclear donor cells and recipient oocytes are recovered from phylogenetically non-consanguineous species (i.e., domestic goat and domestic pig, respectively). Xenogeneic (heterologous) mitochondria that have been inherited from caprine nuclear donor cells and porcine recipient oocytes coexist during preimplantation development of interspecies (goat→pig) cloned embryos, leading to intracellular mtDNA heteroplasmy at the blastocyst stage.

#### **4. Epigenetic Reprogramming of Telomeres in Chromosomes Inherited from Somatic Cell Nuclei throughout Development of Cloned Embryos, Fetuses, and Progeny**

One of the essential prerequisites for epigenetic reprogramming of the cellular memory dependent on somatic cell-derived nuclear genome (nuclear DNA; nDNA) in the ontogenesis of mammals produced by SCNT is the structural–functional rearrangement of nuclear chromatin. The latter is associated with conformational changes in the length of terminal ends of chromosomes known as telomeres [178–182]. In turn, epigenomic biochemical alterations within telomeric chromatin are related to the biocatalytic activity of the telomerase enzyme [3,183–185]. One unresolved problem is the "epigenetic age" of cloned animals, which seems to be correlated to the length of terminal DNA fragments, i.e., the telomeres [186–189]. The telomeres are deoxyribonucleoprotein structures involved in the stabilization of the structure and conformation of nuclear chromatin during the division period of the mitotic cell cycle. This is necessary for the replication of mutation-free genomic DNA and karyokinetic segregation of chromosomes [190–192]. The replication of linear DNA in eukaryotic nuclear chromatin encounters the problem that the 50 -end of the lagging strand cannot replicate, as there is no space for the replication initiating RNA primer. An RNA primer is synthesized on the lagging strand template by primase or RNA polymerase, whose role is played by DNA polymerase α. This creates the risk that somatic cell chromosomes will shorten with every replication round, thus losing genetic information. In mammalian somatic cells, the classical α isoform of DNA polymerase is not capable of semiconservative replication of the 50 -end synthesized in fragments of the DNA chain, whose replication is delayed in relation to the 30 -end of the continuously copied leading strand [182,193]. As a result, in each cell division cycle, unreplicated telomere DNA sequences are gradually lost. For this reason, telomere length is a specific "physiological mitotic clock" of the cell. The shortening of chromosome telomeric regions is positively correlated with the number of cell divisions. Therefore, when the telomere length reaches a critical restriction/control point in a karyokinetically active somatic cell, this is signalized by the loss of nuclear chromatin stability, which is epigenetically programmed in the spatial structure/configuration and telomere functions. This is also signalized by triggering replicative senescence in the cell [187,194–196]. The characteristics of cells that undergo progressive replicative senescence include a considerable increase in diameter and a flattened shape caused by a drastic increase in cytosol volume. All of the above-mentioned epigenetic, genetic, physiological, morphological, and ultrastructural transformations, which occur in aging cells, lead in the first place to a rapid slowdown of both intracellular anabolic processes and the kinetics of mitotic divisions. At a later stage, these transformations bring about the irreversible inhibition of metabolic and proliferative activity. As a consequence of single doubling in the population of mammalian adult dermal fibroblasts cultured in vitro, telomere length decreases by about 48 DNA nucleotide pairs [180,183,188,197,198].

Telomerase is a ribonucleoprotein enzyme complex that displays the total activities of RNA reverse transcriptase and DNA integrase only in germ and embryonic cells, while its partial activity is observed in fetal somatic cells undergoing tissue-specific cytodifferentiation. However, the biocatalytic activity of this enzyme completely ceases in terminally differentiated somatic cells of adult specimens [184,185,193,199]. The function of telomerase is to restore the primary length of DNA telomeres by reverse transcription of its own RNA template. This gives rise to the de novo synthesis (reduplication) of tandem repeats within noncoding telomere DNA sequences (50 -TTAGGG-30 ) that were lost as a result of terminating either consecutive mitotic and meiotic divisions of gametogenic (germinal) cells or mitotic cycles of blastomeres, leading to consecutive cleavage divisions of embryos. In the last phase of semiconservative DNA replication, the 30 -end of the leading strand extends beyond the 50 -end of the lagging strand. Telomerase contains an RNA molecule that is partially complementary to the tandem repeat of the short 50 -TTAGGG-30 sequence at the 30 -end of the leading DNA strand, thus elongating the leading strand of telomeric DNA region using RNA as the template. Next, the enzyme detaches and

binds to a new telomeric end to extend the leading DNA strand. The extension process may occur hundreds of times before telomerase finally dissociates. Then, the extended, replicated leading strand serves as a template for replication of the 50 -end of the lagging strand that is catalyzed by DNA polymerase α. These two processes, where the 50 -ends of DNA are shortened during basic semiconservative replication and subsequently elongated due to telomerase activity, are mutually balanced, whereby the total chromosomal length remains more or less the same [180,192,199,200]. In contrast, a lack of telomerase activity and, as a consequence, a lack of elongating the temporally and spatially restricted length of nuclear DNA telomeric sequences are epigenomically determined factors specific for adult somatic cells that provide a source of nuclear donors for the SCNT procedure. These factors limit the survival rate, proliferative activity, and the number of division cycles of a cell before the cell reaches the critical point of the maximum telomere shortening. The latter is simultaneously the mitotic control point that signals the initiation and irreversibility of the replicative senescence of terminally differentiated somatic cells [188,190,195,201].

The problem of telomere shortening/attrition and replicative senescence of somatic cells was observed in chromosomes of Dolly the sheep, the first cloned mammal [114,178,189,196]. The telomeres in the chromosomes of Dolly the cloned ewe (at the age of 3 years) were much shorter than the telomeres in the chromosomes of control animals, which were of the same age and were born through natural reproduction. Moreover, the telomere length in Dolly's chromosomes was similar to that in the chromosomes of a 6-year-old sheep, which was used as a donor of somatic cells for the cloning procedure. At the time of molecular analysis of telomeres, Dolly was 3 years old, and her epigenetic age corresponded to the actual age of a 9-year-old sheep. Put differently, Dolly's somatic cells were epigenetically older by 6 years than herself. Born on 5 July 1996, Dolly the sheep lived above 6.5 years and was euthanized on 14 February 2003 after being diagnosed with a malignant lung cancer known as Jaagsiekte (ovine pulmonary adenocarcinoma). The etiologic agent of this chronic, contagious, and fatal lung cancer in sheep is Jaagsiekte sheep retrovirus (JSRV), which is responsible for the oncogenic transformation of bronchial exocrine epithelial cells, i.e., type II pneumocytes and bronchiolar club (Clara) cells. By 2000, Dolly produced a total of 6 lambs (including twins and triplets). Therefore, the cloned ewe was reproductively sound and displayed high fertility and prolificacy, which means that her reproductive capacity upon reaching sexual and breeding maturity was not impaired. However, in 2001, the hind legs of 5-year-old Dolly exhibited the first symptoms of an autoimmune chronic degenerative joint disease (osteoarthritis), namely rheumatoid arthritis. It should be noted that this disease is relatively frequent in different breeds of sheep, but generally, it does not affect animals younger than 10 years of age. Two questions arise: Could Dolly live 6–9 years less than the expected lifespan of 12–15 years (which is the average lifespan of Finn Dorset sheep, represented by the somatic cell donor ewe in the SCNT procedure)? As a result of somatic cell cloning, did she exhibit rapidly progressing symptoms of premature (anatomical and physiological) aging of the entire body or of some of its parts, tissues, and organs? The results of experiments performed to determine the telomeric age of Dolly the sheep suggest that animals cloned by transferring adult somatic cell nucleus into the enucleated oocyte are epigenetically compromised. For this reason, they have a genetic age of a specimen playing the role of somatic cell donor for SCNT. This means that at birth, they are epigenetically and genetically much older than their real-time birth date [178,184,189,196]. However, the evidence for cloned sheep was not reflected in the studies focused on the analyses of chromosomes isolated from somatic cells derived from cloned cattle. A study by Lanza et al. [194] on the chromosomes of cloned calves produced by using long-term cultured fibroblast cells for SCNT showed that the telomere length of these young animals is even slightly greater than that of control animals, despite the fact that the chromosomes of nuclear donor cells were almost completely depleted of telomeres. These analyses confirmed that the terminal ends of chromosomes are efficiently resynthesized in blastomeres of bovine cloned embryos, with a contribution from highly active telomerases. Analogously, Tian et al. [179] demonstrated that telomere length in chromosomes of four live (about 15.4 kbp) and six dead cloned calves (about 15.9 kbp) that had been generated using dermal fibroblast cells or cumulus cells derived from a 13-year-old cow not only did not differ

considerably from the telomere length characteristic of control chromosomes (about 14.7 kbp) but also significantly exceeded (by about 3–3.5 kbp) the telomere length in chromosomes of the aging cow (about 12.4 kbp), which served as the donor of somatic cells for SCNT-based cloning. Finally, Kato et al. [202] provided evidence that in cloned cattle, the telomere length is shortened only in the tissues matching those from the biopsy specimens of which the primary cell cultures were established. In turn, the latter provided the somatic cell lines that were nuclear donors used for SCNT procedures.

Telomere length in chromosomes of the dermal fibroblast cells originating from six transgenic cloned pigs matched the telomere length of the chromosomes of dermal fibroblast cells originating from control animals of the same age and produced by natural reproduction. In turn, two cloned piglets that died 3 to 7 days after birth displayed the same length of terminal ends of chromosomes as the telomeres of chromosomes in fetuses at the third trimester of pregnancy [185]. The terminal restriction fragment (TRF) assay of genomic DNA isolated from the cells stemming from the biopsy specimens retrieved from different organs/tissues of cloned fetuses (gonads, heart, liver, lungs, kidneys, and skin) has confirmed that telomere length in chromosomes remains constant in all the cell lines arising from cytodifferentiation that takes place throughout fetogenesis. The reason for this is the high efficiency of restoring the primary length of terminal chromosome ends via the active telomerase isoform throughout the interphase replication cycle of nuclear DNA in differentiating somatic cells that occupy new tissue niches and are engaged in multi-stage histo- and organogenesis processes. During the postnatal period, telomeres are gradually shortened with each mitotic division of somatic cells, and the reduction of telomere length is tissue-specific. This reflects inhibition of the biocatalytic activity of telomerase in differentiated lines of somatic cells derived from skin tissue explants and various internal organs harvested from gilts and boars both before and after attainment of sexual maturity [180,181,185,192,198].

#### **5. Comprehensive Summary and Future Goals**

Cloning by SCNT is currently used in assisted reproductive technologies (ARTs) of many mammalian species, including various species of farm animals. The application of this technology in experimental embryology and in molecular population genetics is of great importance for livestock breeding.

Somatic cell cloning as a method of asexual reproduction offers the opportunity for production and/or multiplication of monogenetic and monosexual progeny of high breeding worth, whose genotypic and phenotypic identity with progenitor donor of transcriptional mitochondrial and nuclear apparatus of the somatic cell only concerns genomic DNA. Animals produced by SCNT differ in phenotypic traits determined by the random segregation of oocyte-derived/maternal and somatic cell-derived/somatogenic mitochondrial genome (mtDNA) as a result of cytoplasmic (extranuclear) inheritance of genetic material [106,133,159,161]. Nevertheless, the particularly high application value of somatic cell cloning technology is related to the possibility of generating genotypically and phenotypically identical transgenic animals, i.e., animals with transformed nuclear genomes that are valuable due to the expression product of modified genes [4,28,38,203]. The yield of recombinant transgenic protein synthesis by genetically transformed cloned specimens is, to a certain extent, dependent on the effect of heteroplasmic sources of mitochondrial genotype (mitotype) on the transcriptional activity profile of modified nuclear DNA genes. This correlation may be negative with a high coefficient of heritability and regressive repeatability of a given quantitative and qualitative trait resulting from the transgenization of a breeding herd [2,133,158]. Therefore, an important problem in the production and multiplication of transgenic cloned specimens (the so-called clonal founder animals) is to generate offspring with an identical mitochondrial genome. These offspring carry only homoplasmic copies of mtDNA derived either from recipient oocytes or from somatic donor cells of genetically modified nuclei. Not without significance is the effect of inheritance of extranuclear genetic information that is accumulated in mitochondrial reservoirs of both somatic (somatogenic)

and germinal (gametogenic) cell lines on the transcriptional activity of quantitative trait loci (QTLs). The latter encompass loci for such traits of transgenic cloned specimens as reproductive traits (e.g., fertility and prolificacy), productive traits, including meatiness (e.g., loin eye area, contents of striated muscle tissue, intramuscular and intermuscular connective tissue, adipose tissue in different carcass, and half-carcass cuts) and milk yield traits (e.g., volume of milk synthesis, and milk secretion and ejection per day and per lactation period) [2,4,106,108,136,157,159,203]. In turn, genetic determinants of prolificacy or fertility traits from the heteroplasmic or homoplasmic pattern of mitochondrial genome segregation may influence the processes of intergenerational transmission of the transgene in germ cell lines of the descendant generations of cloned animals with the transformed nuclear genotype. On the one hand, a negative or positive genetic correlation between milk yield or dressing percentage traits (inherited with genomic DNA) and the transcriptional activity profile of mitochondrial DNA genes may be responsible for different extents or patterns of tissue-specific or organ-specific expression of xenogeneic (e.g., human) gene constructs in transgenic cloned animals. The expression extents or patterns of these gene constructs may be characterized by the inhibition or onset of their transcriptional suppression. On the other hand, the above-mentioned negative or positive correlation may also affect the expression profile of xenogeneic gene constructs (transgenes) in different cells, tissues, and organs of genetically modified cloned specimens. This profile of transcriptional activity of the transgenes integrated with the nuclear genome may be homogenous or heterogeneous, resulting in the induction or absence of transgenic mosaicism/chimerism in cloned animals [4,5,136,158,168,169]. The xenogeneic expressive gene constructs that have been incorporated into genomic DNA of cells localized in different tissues and organs of transgenic cloned animals can encode, for example, recombinant human therapeutic proteins. The synthesis and exo- or endocrine secretion of these proteins can be targeted at secretory cells of the mammary gland or smooth and striated muscle tissue found in all the corporeal organs, organ systems, and parts of farm animals [35,139,204–207].

Intergenomic communication between mitochondrial DNA and the transgene stably integrated with nuclear DNA may also create differences in the efficiency of transgenesis, which induces targeted mutagenesis, i.e., monoallelic deletion or the insertional inactivation of the gene coding for myostatin. Myostatin is a muscle-tissue-specific hormonal protein that paracrinally inhibits the gain (hypetrophy and hyperplasia) of skeletal and smooth muscles [18,208]. The presence of one or two knockout alleles of the myostatin gene or the presence of one or two posttranscriptionally silenced mRNA copies encoded by the myostatin gene in heterozygous or homozygous transgenic cloned beef cattle increases meatiness in cows and bulls. This results from the hypertrophy and hyperplasia of not only striated but also smooth muscle tissue [18,204].

The attractiveness of SCNT-based cloning of transgenic mammals, including various species of domesticated animals, is decided by the applicability of the hormonal or enzymatic product of the modified gene expression. This applicability first of all determines the scale and scope of the research. Although the first cloned mammal was a sheep, research targeted at the somatic cell cloning of other livestock species had a much wider span. The mammary glands (udders) of transgenic cloned cows [11,12,14,206,209,210], transgenic cloned sheep [23,24], and transgenic cloned goats [17,19] may become live bioreactors for producing humanized milk, easy-to-digest milk, or milk containing recombinant human therapeutic proteins (biopharmaceuticals or nutraceuticals). The latter may find clinical application in the treatment of patients afflicted with genetically determined diseases [12,211–213].

Compared to other ARTs in mammals (including livestock species), the efficiency of somatic cell cloning in domesticated animals, which is measured by the percentage of offspring born in relation to the number of reconstructed oocytes, remains low and oscillates between 0.3% and 2% on average. Nonetheless, the biotechnological possibilities of the somatic cell cloning in different mammalian species is far ahead of our understanding of the biological determinants, in particular the molecular and epigenetic aspects, of this

method [108,132,192,203]. Yet, the biological foundations that have been laid for embryonic genome engineering of domesticated animals, especially over the last 24 years, made feasible the development of an innovative technology of in vitro embryo production using the somatic cell cloning procedure, which may meet the requirements for application in laboratories or, in some cases, only for limited practical purposes [127,214,215]. The assisted reproductive technology that encompasses SCNT could be used on a larger practical scale only after the efficiency of somatic cell cloning in various mammalian species, including livestock, is increased to match the efficiency of in vitro fertilization (IVF) or artificial insemination (AI) as part of multiple ovulation and embryo transfer (MOET) programs in cattle. However, due to the relatively high incidence of lethal or sublethal developmental anomalies or anatomo-histological defects in cloned fetuses and progeny, it is not possible to use the somatic cell cloning of farm animals on a commercial scale, at least at the present level of sophistication of the relevant research performed in Europe and the world. Furthermore, it is also worth noting that the elaboration and optimization of efficient approaches applied to cryopreserving nuclear donor somatic cells, nuclear-transferred oocytes reconstructed with somatic cells, and somatic cell-cloned embryos appear to be important milestones that can help cryogenically protect these valuable types of biological materials. This can bring the investigators closer to the perspective of progression in the outcome of producing mammalian SCNT progeny. In turn, future large-scale attempts undertaken to more successfully generate mammalian cloned offspring can expedite their practical use for the purposes of not only agricultural, transgenic, biotechnological, biomedical, and biopharmaceutical research fields but also ex situ conservation of biological diversity in different anthropogenic and unspoiled natural ecosystems.

To sum up, it seems that after making the transition from basic to applied research, the techniques for intra- and interspecies somatic cell cloning of mammals could contribute to (1) the conservation of genetic resources and the establishment of the genetic reserves of threatened mammalian species and breeds, (2) the restoration and multiplication of the subpopulations of endangered or vulnerable wild and domesticated species of mammals in order to maintain biodiversity and to increase the level of intra-population and interspecimen genetic variability, and (3) revival ("resurrection") and reintroduction into the wild of extinct, free-living species of mammals. Moreover, the practically applied research into the cloning of domesticated animals could serve to achieve other tangible benefits, including (4) the improvement of the breeding (genetic) and productive value of different farm animal breeds, e.g., increasing their milk and meat yields and reproductive ability (prolificacy and fertility), and (5) the implementation of basic research into interdisciplinary sciences aimed at the generation of animal biotechnological (transgenic) products for the biomedical, biopharmaceutical, nutraceutical, and food technology industries. One classic example of this is the permanent and highly heritable targeted transgenization of the mammary glands of domesticated species of small and large ruminants (i.e., sheep, goats, and cattle, respectively) and their use as animal bioreactors for producing humanized milk or milk containing recombinant human therapeutic proteins such as biopharmaceuticals and nutraceuticals.

**Author Contributions:** Conceptualization, M.S. (Marcin Samiec); Writing the article—original draft, M.S. (Marcin Samiec) and M.S. (Maria Skrzyszowska); Writing the article—review and editing, M.S. (Marcin Samiec); Supervision and funding acquisition, M.S. (Marcin Samiec); Graphic documentation, M.S. (Marcin Samiec). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Ministry of Science and Higher Education in Poland as a statutory activity No. 04-19-05-00.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

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

**Maria Skrzyszowska \*,† and Marcin Samiec \*,†**

Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Krakowska 1 Street, 32-083 Balice n. Kraków, Poland

**\*** Correspondence<EMAIL_ADDRESS>(M.S<EMAIL_ADDRESS>(M.S.)

† These authors contributed equally to the preparation of this paper.

**Abstract:** The domestic goat (*Capra aegagrus hircus*), a mammalian species with high genetic merit for production of milk and meat, can be a tremendously valuable tool for transgenic research. This research is focused on the production and multiplication of genetically engineered or genomeedited cloned specimens by applying somatic cell nuclear transfer (SCNT), which is a dynamically developing assisted reproductive technology (ART). The efficiency of generating the SCNT-derived embryos, conceptuses, and progeny in goats was found to be determined by a variety of factors controlling the biological, molecular, and epigenetic events. On the one hand, the pivotal objective of our paper was to demonstrate the progress and the state-of-the-art achievements related to the innovative and highly efficient solutions used for the creation of transgenic cloned does and bucks. On the other hand, this review seeks to highlight not only current goals and obstacles but also future challenges to be faced by the approaches applied to propagate genetically modified SCNTderived goats for the purposes of pharmacology, biomedicine, nutritional biotechnology, the agri-food industry, and modern livestock breeding.

**Keywords:** domestic goat; somatic cell cloning; SCNT-derived embryo; genetically engineered specimen; gene targeting; genome editing; biopharmacy; biomedicine; nutri-biotechnology

#### **1. Introduction**

One of the most rapidly developing strategies for reproductive biotechnology in mammals, including farm livestock species, is cloning by somatic cell nuclear transfer (SCNT) (Figure 1).

It is beyond any doubt that the attractiveness of cloning techniques results from their potential to generate and multiply transgenic animals, which are valuable due to the expression of modified genes (Figure 1). Furthermore, this attractiveness also depends, to a lesser degree, on the possibility to replicate individuals with excellent, highly heritable breeding (genetic) and performance traits, which may shorten the generation interval and increase the rate of breeding progress. However, the aforementioned areas of research are being explored on a limited scale due to the high costs associated with the cloning procedure resulting from the low efficiency of the method. It is beyond any doubt that the widespread use of cloning methods will be possible once efficacy and repeatable results are guaranteed [1–4].

The main reason for low pre- and postimplantation developmental potential and poor quality of SCNT-derived embryos is the abnormal adaptation of the transferred somatic cell nuclei to the biochemical conditions of the oocyte cytoplasmic microenvironment, i.e., their incomplete or improper remodeling and reprogramming in the cytoplasm of nucleartransferred oocytes. The latter also gives rise to the relatively high incidence of congenital malformations (anatomo-, histo-, and physiopathological changes) in cloned fetuses and

**Citation:** Skrzyszowska, M.; Samiec, M. Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine. *Int. J. Mol. Sci.* **2021**, *22*, 7490. https://doi.org/10.3390/ ijms22147490

Academic Editor: Sato Masahiro

Received: 11 June 2021 Accepted: 9 July 2021 Published: 13 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

offspring. This calls for studies aimed at the precise determination of the conditions that facilitate epigenetic reprogramming in the nuclear donor cell genome during the preand postimplantation development of SCNT-generated embryos and fetuses of different mammalian species, including the domestic goat [5–10]. 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 [11–15]. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 2 of 16

**Figure 1.** Generation of transgenic cloned goats by somatic cell nuclear transfer (SCNT). **Figure 1.** Generation of transgenic cloned goats by somatic cell nuclear transfer (SCNT).

#### The main reason for low pre- and postimplantation developmental potential and poor quality of SCNT-derived embryos is the abnormal adaptation of the transferred **2. Key Issues Related to Biological, Molecular, and Epigenetic Determinants Affecting the Efficacy of Somatic Cell Cloning in Goats**

somatic cell nuclei to the biochemical conditions of the oocyte cytoplasmic microenvironment, i.e., their incomplete or improper remodeling and reprogramming in the cytoplasm of nuclear-transferred oocytes. The latter also gives rise to the relatively high incidence of congenital malformations (anatomo-, histo-, and physiopathological changes) in cloned fetuses and offspring. 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 The provenance of somatic cells is a factor that can have a significant impact on cloning efficiency in goats. Relatively few types of nuclear donor cells have been tested for their suitability for the production of cloned embryos, fetuses, and/or offspring in this livestock species. Those that have been tested include cells stemming from several types of tissues collected from both caprine fetuses and adult animals of both sexes and of different ages. Among the nuclear donor cells (NDCs) used for SCNT procedures, mention should be made of: (1) in vitro cultured (transgenic or nontransgenic) fetal dermal fibroblasts [12,16–22];

and fetuses of different mammalian species, including the domestic goat [5–10]. Promising results were achieved by investigations that focused on the use of extrinsic nonselective agents for stimulating the epigenetically regulated transcriptional activity of ge(2) juvenile and adult dermal fibroblasts [14,23–26]; (3) mural granulosa cells isolated from antral ovarian follicles [18,27]; (4) cumulus oophorus cells [18,27,28].

Special consideration should be given to the use of pituicytes, i.e., endocrine cells originating from the anterior pituitary (also known as adenohypophysis) of postpubertal bucks, as a source of nuclear donors for SCNT in goats [29]. To date, this cerebral tissue-specific type of endocrine cell, which synthesizes and secretes tropic hormones, has not been used for SCNT in other species of farm and laboratory animals. It has been postulated, however, that artificial (ectopic) control of metabolic and secretory activities of the endocrine compartment in all lobes of the pituitary gland of livestock species might be feasible through genetic modification (transfection) of pituicytes at the in vitro culture level. In turn, utilizing genetically transformed pituitary-derived glandular cells, which are characterized by inducible expression of recombinant human hormonal proteins or polypeptides, for the generation of transgenic specimens of different mammalian species by somatic cell cloning opens up a variety of new application opportunities. The latter encompass the production of transgenic animal bioreactors, which provide xenogeneic (human) tropic hormones in cytosol extracts (homogenates) of pituicytes or in blood plasma. These hormones are indispensable for the clinical application of therapies for many human monogenic diseases, which induce endocrine-mediated congenital malformations. The transfer of caprine SCNT embryos that had been reconstructed with pituicytes into the reproductive tract of hormonally synchronized recipient females resulted in the birth of a cloned male kid. The results of these experiments confirmed that even the nuclear genome of terminally differentiated somatic cells such as pituicytes can successfully undergo the complete processes of epigenetic remodeling and reprogramming in pre- and postimplantation cloned goat embryos [29].

Deng et al. [30] examined the methylation profile and expression level of the *Xist* (X-inactive specific transcript) gene in the cells of SCNT embryos and in ear fibroblast cells, lung-derived cells, and cerebral cells collected from deceased cloned goats. The methylation profile observed for the *Xist* gene, which is transcribed into a noncoding mRNA molecule, i.e., a transcript that does not exhibit translational activity, was higher in 8-blastomere-stage SCNT embryos as compared to their in vitro-fertilized embryo counterparts generated by intracytoplasmic sperm injection (ICSI). Moreover, an increased methylation profile of the *Xist* gene was observed in the cells stemming from explants representing tissues/organs such as conchal skin, lungs, and brain, isolated postmortem from dead 3-day-old cloned female kids in relation to naturally bred specimens. While for ear skin-derived tissue bioptates originating from live cloned does, and for lung and brain tissue samples retrieved from dead cloned kids, the methylation profile of 50 -cytidine-3 0 -monophopshate-50 -guanosine-30 (CpG) islands within the differentially methylated regions/imprinting control regions (DMRs/ICRs) of the *Xist* gene remained unchanged. Therefore, the transcriptional activity of the *Xist* gene diminished remarkably in the lungs and brain of dead cloned does, resulting in a lack of inactivation recognized for one of the X chromosomes (either of paternal or of maternal origin) in the cells of the previously indicated organs. In turn, a significant increase in *Xist* gene expression was shown in the ear-derived cutaneous fibroblast cells of live cloned does. This contributed to the normal inactivation of one of the two X chromosomes in these specimens. It is evident from this study that an increased incidence of hypermethylation and transcriptional suppression of the *Xist* gene, and thus no inactivation of one of the two X chromosomes, or in other words, active initiation of enhanced transcriptional activity (i.e., biallelic overexpression) of the genes localized in the loci of the paternal and maternal X chromosomes occurred in caprine SCNT-derived female fetuses. For these reasons, the aforementioned processes, which were also identified in the tissue explants recovered from 3-day-old dead cloned does, are found to arise from incomplete and aberrant reprogramming and were highlighted as a result of the epigenetically determined transcriptional activity of the somatic cell nuclear genome in cloned goat embryos [30].

Incomplete or incorrect epigenetic reprogramming of epigenetic memory, which is encoded in extragenic covalent modifications of the somatic cell nuclear genome, was found to be one of the main factors decreasing the efficiency of somatic cell cloning in mammals, including the domestic goat. Reductions in this efficiency are reflected in the weakened in vitro and/or in vivo developmental potential of SCNT-derived embryos [7,8,31]. Methylation of cytosine residues in CpG islands/dinucleotides is a widely explored/recognized modification of the somatic cell nuclear genome in cloned embryos [6,9,10,32]. Han et al. [33] demonstrated that enzymatic activity of ten-eleven translocation methylcytosine dioxygenase 3 (TET3) is a key molecular mechanism underlying active DNA demethylation in preimplantation goat embryos created by somatic cell cloning. Knocking out the *TET3* gene led to the inhibition of active (i.e., DNA replicationindependent) demethylation of 5-methylcytosine (5-mC) residues in 2-blastomere-stage cloned goat embryos. As a consequence, this brought about the downregulation of the expression of the pluripotency-related *Nanog* gene in the inner cell mass (ICM) compartment of the generated blastocysts. In turn, overexpression of the *TET3* gene that had been induced by transgenization of in vitro cultured somatic cells resulted in: (1) abundant demethylation of DNA 5-mC residues; (2) declined quantitative profile of 5-mC moieties; (3) increased incidence of 5-hydroxymethylcytosine residues; (4) intensified transcriptional activity of crucial pluripotency-related genes. Furthermore, the use of genetically transformed somatic cells displaying overexpression of the *TET3* gene—as nuclear donors for the reconstruction of caprine enucleated oocytes—contributed to an enhancement in the extent of active demethylation of 5-mC residues within somatic cell-inherited nuclear DNA. The latter perpetuated hypomethylation of the somatic cell-derived genome in cleaved SCNT embryos, subsequently triggering remarkable improvements in their in vitro and in vivo developmental capabilities. It follows that overexpression of the *TET3* gene in NDCs significantly ameliorates the efficacy of somatic cell cloning in goats.

The developmental potential of the mammalian SCNT embryos, including their caprine representatives, which inherit the somatic cell nuclear genome as a result of the reconstruction of enucleated oocytes, is highly dependent on the level of epigenetic modifications within DNA and chromatin-derived histones of the NDCs undergoing longterm in vitro culture [34–36]. One of the strategies to reverse advanced alterations in the pattern of epigenetic covalent modifications within somatic cell nuclei, which encompass rapid DNA methylation and a decrease in the quantitative profile of histone protein acetylation, appears to be the exposure of NDCs, SCNT-derived oocytes, and corresponding embryos to reversible agents inhibiting biocatalytic activity of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs). The use of nonselective or selective promoters of epigenetically determined transcriptional activity of genomic DNA in both in vitro cultured NDCs and cloned embryos is supposed to be an approach that allows for proper reprogramming of somatic cell nuclei [7,12,14]. Exogenously modulating the epigenetic memory profile of genomic DNA appears to contribute to successfully reversing the "transcriptional clock" of a differentiated somatic cell nucleus to the status of a cell nucleus characteristic of a totipotent or pluripotent embryonic cell. As a consequence, such efforts induce the restoration of the expression pattern that is seen in genes that are inevitable in the initiation and progress of the developmental program of SCNT-derived embryos [37,38]. This results in a reduction in the methylation degree of DNA cytosine residues and an increase in the acetylation of nuclear chromatin histone proteins [39,40]. In turn, the previously specified processes were shown to bring about recapitulation and perpetuation of the correct and faithful profiles of transcriptional activities observed both for the genes indispensable to induce and maintain the totipotency/pluripotency states and for the genes encoding enzymes responsible for endogenous epigenetic modifications during pre- and postimplantation embryogenesis. The totipotency/pluripotency-related genes encompass those that encode such proteins as: e.g., octamer-binding transcription factor 3/4 (Oct3/4), the homeobox-containing transcription factor Nanog, whose name stems from the Celtic/Irish mythical word Tír na nÓg (i.e., The Land of the Ever-Young), DNA-

binding proto-oncogenic/oncogenic transcription factor c-Myc, sex-determining region Y (SRY)-box 2 transcription factor (Sox2), Krüppel-like factor 4 (Klf4), reduced expression protein 1 (Rex1), and caudal-type homeobox protein 2 (Cdx2). The epigenetic modifier genes involve those that encode such enzymatic proteins as: e.g., DNA methyltransferase type 1 (DNMT1), DNA methyltransferase type 3a (DNMT3a), DNA methyltransferase type 3b (DNMT3b), histone deacetylase type 1 (HDAC1), histone deacetylase type 2 (HDAC2), histone methyltransferase (HMT), and histone acetyltransferase (HAT) [3,41,42]. In the wake of recent research, innovative and highly efficient methods were developed to modulate the epigenetic memory profile of mammalian SCNT embryos, including their caprine counterparts. These methods are focused on applying exogenous nonselective HDAC inhibitors (such as trichostatin A, valproic acid, and scriptaid) and/or nonselective DNMT inhibitors (such as 5-aza-20 -deoxycytidine) or selective inhibitors lysine K4 demethylases specific for histones H3 within the nucleosomal core of nuclear chromatin (such as trans-2-phenylcyclopropylamine (tranylcypromine; 2-PCPA)). The aforementioned strategies may considerably modify the epigenetically determined reprogramming of the somatic cell nuclear genome in SCNT-derived embryos. The final results of these innovative solutions turn out to be significant enhancements of the pre- and/or postimplantation developmental competence and an improvement in the molecular quality of cloned embryos in mammals, including the domestic goat [12–14,42–44].

#### **3. Species-Specific Advantages of the Goat That Increase the Potential for Its Practical Application in Transgenics, Biopharmacy, Biomedicine, and Biotechnology**

The domestic goat (*Capra aegagrus hircus*), a species with a tremendously high biodiversity of breeds showing relatively high milk and/or meat yield, may serve as an excellent research subject for SCNT-mediated production of transgenic bioreactor specimens. These caprine genetically engineered bioreactors of foreign species-descended (xenogeneic) biopreparations can provide recombinant human therapeutic proteins that are designated as biopharmaceuticals or nutraceuticals (Table 1), together with physiological secretions (e.g., milk) and excreta (e.g., urine). Moreover, somatic cell cloning in this livestock species seems to be a reliable, feasible, and powerful tool for generating and/or multiplying specimens (does and bucks) that display genetically modified parameters of meatiness and intramuscular adipose tissue content (Table 1). Increasing the efficiency of producing purified xenogeneic biopharmaceuticals or bionutraceuticals derived from the mammary glands (udders) of transgenic goats would thus allow them to be phased into the biopharmaceutical industry [24,45]. Another tangible benefit of producing transgenic cloned goats, which appears to be especially valuable for the xenogeneic product of the transgenic expression of exogenous DNA (directed at the mammary glands or resulting in higher meatiness), is the relatively short species-specific generation interval. The latter allows for increasing the rate of genetic progress in the breeding of founder does and bucks [24,25]. Yet another advantage of this small ruminant species is the low susceptibility of dairy and meat goats to infection with pathological prions (PrPSc) that cause scrapie in sheep [27,46,47]. Transgenic goats may serve as optimal bioreactors to produce human therapeutic proteins for various agroeconomic reasons. Compared to the breeding of transgenic cows, these animals are more easily farmed, their natural and biotechnologically assisted reproduction can be more rapidly controlled, and they are much cheaper to keep as compared to large ruminants. Relative to their body size, they have fairly large udders with a predominance of glandular tissue over fibrous parenchyma, which makes this small ruminant species genetically predisposed to a high production potential of colostrum and milk. The possible consequence of these anatomo-physiological advantages in goats is the high performance of transgenic doe herds in terms of lactogenic synthesis and secretion of recombinant human therapeutic proteins (biopharmaceuticals or nutraceuticals) by alveolar mammary epithelial cells. These caprine cells provide udder-derived secretion with a genetically modified qualitative and quantitative composition [24,48,49].

**Table 1.** Targets and effects of genetic modification in transgenic cloned goats.

The first transgenic cloned kids were generated from the nuclear-transferred embryos reconstructed with caprine somatic cells that had been previously transfected in vitro with relatively simple gene constructs. These gene constructs contained no genomic sequences of the regions encoding structural transgenes, but were composed of the exon segments of genes encoding selectable marker proteins, e.g., *PGKneo* fusion genes. The aforementioned fusion genes are comprised of murine phosphoglycerate kinase (*PGK*) promoters and neomycin phosphotransferase (*neo*) genes. The *neo* gene determines resistance to selective aminoglycoside antibiotic designated as geneticin disulphate (G418 sulphate). In the study

by Zou et al. [19], in vitro cultured fetal fibroblasts provided a source of transgenic NDCs for SCNT-based cloning in goats. The transgenic NDCs were created by their transfection with a gene construct that only contained the *neo* gene. The above-mentioned study resulted in the production of five genetically modified kids. In turn, Keefer et al. [17] and Baldassarre et al. [27,46] used the in vitro lipofection approach to transfect fetal fibroblasts with a more complex plasmid gene construct (*CEeGFP*). The *CEeGFP*-fusion gene was composed of: (1) the enhanced green fluorescent protein (*eGFP*)-reporter gene driven by the human elongation factor-1α promoter and cytomegalovirus enhancer and (2) the *neo* gene under the control of the simian virus-40 (*SV-40*) promoter. Following the transfer of genetically transformed cloned embryos into the reproductive tracts of recipient surrogates, one cloned doe showing the expression of the *eGFP*-reporter transgene was produced.

Transgenic animals with the high transcriptional activity profiles (diagnosed in vivo) of a xenogeneic gene may be subsequently multiplied by somatic cell cloning. This is particularly justified when biopharmaceuticals stemming from these animals may find widespread application in the treatment of patients suffering from various single-gene heritable disorders. When transgenic biopharmaceuticals obtain certification for application in humans, somatic cell cloning of genetically modified specimens will allow, at least in theory, for the maintenance of homogeneity of the drugs extracted from natural secretions and excretions (milk, urine) of the successive generations of cloned animals. This technology was successfully used by the American biotechnology company GTC Biotherapeutics (formerly Genzyme Transgenics Corporation), which generated transgenic goats exhibiting monoallelic expression of recombinant human antithrombin III gene (*rhAT*) in their mammary glands (udders). The production of transgenic cloned goats was based on the use of a standard intrapronuclear microinjection into the zygotes of cDNA constructs containing the goat β-casein gene promoter. In the performed experiments, genetically modified fibroblast cell lines were established from fetuses obtained by mating nontransgenic does with a genetically modified founder buck. This buck displayed transcriptional activity of the *rhAT* gene that was directed into the mammary gland (udder). Clonal lines of transgenic fetal fibroblast cells served as a source of nuclear donors in the somatic cell cloning procedure, which resulted in a total of eight genetically engineered SCNT-derived female kids [16,45]. The findings of Cheng et al. [28] represent another example of applying the somatic cell cloning technique for multiplying populations of genetically transformed specimens. In this case, enucleated oocytes were reconstructed by SCNT with the use of in vitro cultured fibroblast cell lines collected from dermal tissue explants of a transgenic goat displaying ubiquitous expression of recombinant human erythropoietin (rhEPO). After surgical transfer of the cloned embryos into the reproductive tracts of hormonally synchronized recipient does, two genetically modified kids were born. The SCNT-derived offspring were characterized by mammary gland-specific expression of xenogeneic rhEPO protein.

#### **4. Transgenic Cloned Goats as Bioreactors That Produce Recombinant Human Therapeutic Proteins**

The nuclear transfer of in vitro-transfected somatic cells increases the probability of producing nonmosaic transgenic offspring, which have an exogenous gene construct incorporated into the primordial germ cell line. Such specimens, which are identified as nonchimeric with regard to the genetic transformation of gametogenic and somatic cells, retain their full capacity to transmit phenotypically and molecularly diagnosed transgene expression to the secretory epithelial cells (lactocytes) in the mammary glands of the next generation of kids [49,55,56]. An outstanding example is found in the findings of Baguisi et al. [45]. High-level expression of the *rhAT* gene detected in the udder lactogenic cells of three cloned does, which were produced from SCNT embryos reconstructed with transgenic fetal fibroblasts, was also reflected in the very high phenotypic value of this genetically modified trait in the milk samples. Over a 33-day lactation induced at 2 months of age, the milk yield of these genetically engineered does reached approximately 160 mL. Additionally, the rhAT concentration in the collected milk was maintained at a level

of as much as 5.8 g/L (20.5 U/mL was observed for the enzymatic activity of purified biopharmaceutical) at day 5 and 3.7 g/L (14.6 U/mL for the biocatalytic activity) by day 9 of lactation. At such high concentrations of recombinant therapeutic proteins in milk, large-sized herds of transgenic goats could easily yield 300 kg of extracted (purified) biopharmaceutical product per year. Combining somatic cell cloning technology with hormonal induction of early lactation in prepubertal transgenic does will shorten the time needed to obtain the transgene expression product by as much as 8 to 9 months from the time of cell line transfection to the secretion of the genetically engineered protein biopreparation into milk [20,45]. On the one hand, the volume of these retrieved milk samples is sufficient for estimating the recombinant protein yield. On the other hand, taking into account even a relatively low quantitative profile of translational activity identified for transgene-transcribed mRNA (as measured by milligram quantities of therapeutic protein per 1 mL of milk), this amount of milk can be used for multiple clinical tests of the pharmacokinetic, hormonal, and enzymatic activity of the produced biopharmaceuticals.

Special consideration should be given to the broad international commercialization of the first biopharmaceutical in 2006–2009, designated as ATryn®, by GTC Biotherapeutics. The basic active biochemical component of this pharmacological biopreparation is rhAT, which was recovered from the milk synthesized and secreted by the udders of transgenic cloned specimens of a large livestock species, the domestic goat [47,57,58]. This is a milestone in the practical, commercial-scale implementation of the first biopharmaceutical product of modern mammalian reproductive biotechnology based on embryonic genome engineering technologies such as transgenesis and somatic cell cloning of farm animals. It is worth pointing out here that ATryn® is the world's first drug to be provided by mammary gland-based bioreactors of genetically engineered cloned goats that exhibit highly efficient and organ-specific mono- or biallelic expression of the *rhAT* transgene. This biopharmaceutical was originally granted a marketing authorization by the European Medicines Agency (EMA) in 2006 for use in the biopharmaceutical and medical sector of the European Union, followed by certification from the United States Food and Drug Administration (FDA or USFDA) in 2009 for marketing in the biopharmaceutical and biomedical sector in the USA and Canada [1,59]. At this stage, ATryn® is widely used in biomedical programs/therapeutic platforms for the treatment of hereditary AT deficiency in hospitalized medical patients [60,61].

Another example of the practical application of the mammary glands of genetically transformed cloned goats as bioreactors to synthesize human therapeutic proteins or socalled humanized milk is found in the study by Zhu et al. [21]. This investigation was aimed at ameliorating allergic reactions and inflammatory responses to β-lactoglobulin (BLG) protein (Table 1). As a major whey protein with potential allergenic effect, BLG occurs in the milk of all even-toed mammals (*Artiodactyla*), including the domestic goat. It has no allergenic properties in human milk. The presence of this protein in caprine milk considerably limits, to a high degree, the consumption of this lactogenesis-derived product despite its high nutritive value and health-promoting benefits. Using conventional homologous recombination, the above-mentioned investigators were the first to functionally inactivate a single copy of the *BLG* gene, either through *BLG* gene knockout or through *hLA* (human α-lactalbumin) gene knock-in into the nuclear genome of in vitro cultured fetal fibroblast cells. These cells subsequently provided a source of nuclear donors for reconstructing enucleated doe oocytes in the somatic cell cloning procedure. The ultimate outcome of this research was the birth of three SCNT-derived kids, among which mono-allelic knockout of the targeted *BLG* gene was confirmed in two specimens (Table 1) [21].

In turn, Yuan et al. [22] used the strategy of somatic cell cloning to generate transgenic goats whose udders were bioreactors that synthesized humanized milk containing pharmaceutical or nutraceutical immune glycoprotein, known as recombinant human lactoferrin (hLF). Genetically transformed fetal fibroblasts provided the source of nuclear donor cells for the reconstruction of enucleated oocytes by somatic cell cloning. The genome of nuclear donor cells had been previously edited by inserting *hLF* cDNA into the *BLG* locus, i.e., by

replacing the *BLG* gene with the *hLF* gene. Editing the nuclear genome of fetal fibroblast cells had been mediated by transcription activator-like effector nucleases (TALENs). The efficiency of the targeted mutagenesis observed for the *BLG* gene oscillated at the level of approximately 10%. The results of investigations by Yuan et al. [22] confirmed that the combination of TALEN-based genome editing with the SCNT strategy gave rise to biallelic inactivation of the *BLG* gene through the knock-in of *hLF* exons into the genomic DNA of cloned goats whose mammary glands were targeted by programmed genetic transformation to produce recombinant hLF (Table 1). Transgenically encoded qualitative and quantitative modification of the biochemical composition of caprine milk that subsequently brought about the production of humanized milk in the udders of SCNT-derived goats appears to ameliorate its allergenicity. Diminishing the capability of the milk provided by genetically engineered bioreactors to trigger acute allergic reactions in humans is simultaneously reflected in enriching the goat milk with the valuable multipotent protein designated as LF. This immune glycoprotein, apart from physiologically regulating the dynamic homeostasis of the metabolism of iron cations, is characterized by several other desirable immunotherapeutic properties, including antimicrobial (antibacterial, mycostatic, and antiviral), immunomodulatory, anti-inflammatory, and anticancer abilities (Table 1) [22,24,56].

It is also noteworthy that Zhang et al. [50] reported the effective integration of the recombinant *hLF* gene with the xenogeneic host genome as a result of genetically transforming the caprine fetal fibroblast cells under in vitro culture conditions. The genetically transformed fetal fibroblasts were subsequently used to generate transgenic kids (does) with the *hLF* gene in ear skin tissue samples by somatic cell cloning (Table 1). Out of the six transgenic cloned kids produced, three does died during the perinatal period due to severe bronchopulmonary dysplasia in underdeveloped lungs and acute hypoxemic respiratory failure. The epigenetic analysis of tissue explants collected postmortem from the lungs of perished transgenic does revealed hypermethylation of CpG islands/dinucleotides within the DMR/ICR domain of the gene encoding insulin-like growth factor 2 receptor (IGF2R). For that reason, the maternal allele of the *IGF2R* gene was found to be transcriptionally overactive/upregulated due to enhanced methylation of cytosine residues in the DMR/ICR-associated intron sequence, while its paternal counterpart was shown to be transcriptionally silenced due to the occurrence of parent-of-origin and allele-specific methylation imprint. As a result, the overexpression of mRNA transcribed by the maternal allele of the *IGF2R* gene that had undergone aberrant genomic imprinting was identified in the cell samples of postmortem isolated lung tissue explants stemming from the cloned kids [50].

In summary, imprinted genes are an important epigenomic regulator of anatomohistological growth and development and physiological maturation of the lungs. In turn, aberrant or incomplete reprogramming of the epigenetically determined transcriptional activity of DNA, which underlies abnormal (i.e., increased) methylation of cytosine moieties within DMR/ICR-related intron sequences of the imprinted maternal allele of the *IGF2R* gene, determines the monoallelic overexpression of this gene exprimed from the maternal genome in the lungs of cloned fetuses. The latter appears to be one of the main lethal factors positively correlated with etiopathogenesis of lung hypoplasia and acute pulmonary insufficiency in neonatal transgenic cloned kids [50].

#### **5. Transgenic Cloned Goats as a Source of Valuable Meat for Humans**

Attempts to create and multiply transgenic cloned goats may provide a research basis for the SCNT-mediated generation of genetically engineered specimens (bucks and does) that exhibit genotypic and phenotypic modifications related to increased carcass meatiness and decreased intramuscular fatness. These animals could serve as a valuable research model for expanding our knowledge of the importance of myostatin, e.g., in the context of the quality and taste of the meat from individuals displaying superior gains in muscle tissue and myofiber size. Myostatin, encoded by the *MSTN* gene, is a hormonal inhibitory

polypeptide that inhibits/downregulates the growth, differentiation, maturation, and development of skeletal muscles in mammals. Research shows that functional inactivation of the *MSTN* gene using gene targeting (targeted mutagenesis) or genome editing techniques contributes to increase skeletal muscle mass while diminishing the content of intramuscular adipose tissue and reducing genetically determined or diet-induced obesity. This has the beneficial effects of increased meat yield, fattening performance, and dressing percentage in genetically modified males and females generated by SCNT-based cloning. These effects are evoked by cellular hyperplasia (proliferation) and hypertrophy (enlargement) of striated muscle tissue (Table 1). The hyperplasia and hypertrophy of striated muscle cells (i.e., skeletal sarcocytes known as syncytial myocytes) are synergistically triggered as a result of the following:

It is beyond any doubt that SCNT-derived goats that are characterized by *MSTN* gene silencing (Table 1) [25] represent a powerful, reliable, and feasible tool for investigations targeted at nutritional physiology, food technology, dietetics, nutrigenomics, nutriepigenomics, nutritranscriptomics, nutriproteomics, and human nutrition metabolomics and metabonomics. Myostatin gene knockout (*MSTN*-KO) in goats (Table 1) [25,62,64] and sheep [63,65] was investigated in several research centers. However, as a result of the low efficacy of homologous recombination (HR)-mediated targeted mutagenesis, shorthairpin RNA-mediated gene targeting, and zinc-finger nuclease (ZFN)-mediated genome editing, only a few studies resulted in successful *MSTN* gene knockdown in ex vivo expanded caprine juvenile cutaneous fibroblasts [25], ovine fetal myoblasts [63], or ovine fetal cutaneous fibroblasts [65].

In recent years, other noteworthy solutions have been applied to genome editing and have been subsequently adapted to ARTs. These were then applied in combination to cloning goats using SCNT. These solutions are aimed at strategies based on the use of TAL-ENs or the clustered regularly interspaced short palindromic repeat/CRISPR-associated endonuclease type 9 (CRISPR/Cas9)-assisted system. The progress achieved in developing and optimizing the techniques of targeted knockout of specific gene loci is promising. Therefore, these techniques are increasingly utilized for precise genome editing, allowing for the genome of mammals, including the domestic goat, to be modified with relative ease [66–71]. Ni et al. [53] were the first to demonstrate that CRISPR/Cas9-mediated genome editing can induce accurate mono- or biallelic mutations in the *MSTN* gene of caprine fetal fibroblast cells. The clonal lines of these NDCs that exhibit biallelic *MSTN*-KO were used in a SCNT procedure, resulting in three live-born cloned kids, all of which carried a biallelic mutation in the form of double inactivation of the *MSTN* gene's loci (Table 1).

In turn, Yu et al. [54] showed that TALEN-based genetic transformation leads to the successful inhibition of *MSTN* gene expression in gene-edited cloned goats (Table 1). Moreover, the outcome of both TALEN- and CRISPR/Cas9-mediated systems that were applied to edit the nuclear genome of SCNT-derived Alpas breed cashmere goats (Table 1) was evaluated by Zhang et al. [52]. The efficiency of triggering *MSTN*-KO was compared at many levels of pre- and postimplantation development of transgenic cloned embryos. The rates of both electro-transfecting/electroporating the somatic cells and cutting exon 1 within the *MSTN* gene were found to be higher for the CRISPR/Cas9-assisted strategy of genome

editing than for its TALEN-mediated counterpart. Nevertheless, the genome-wide offtarget effects were shown to be more frequent for the CRISPR/Cas9-mediated system than for the TALEN-mediated system. Furthermore, for CRISPR/Cas9-based genome editing, the incidence of effectively inducing targeted biallelic mutagenesis of the *MSTN* gene increased over eight times as compared to TALEN-based genome editing. In turn, caprine SCNT embryos that had been reconstructed with TALEN-mediated transgenic NDCs reached the 8-blastomere stage more quickly and their cleavage activity was significantly higher as compared to SCNT embryos derived from CRISPR/Cas9-mediated gene-edited NDCs. However, cloned kids were produced, following the surgical transfer of SCNT embryos into recipient does, that stemmed from NDCs that were genetically modified only using the CRISPR/Cas9-assisted technique (Table 1). This ultimately suggests that the high yield of generating targeted modifications of the *MSTN* gene (*MSTN*-KO) was achieved using CRISPR/Cas9-mediated genome editing [52].

To summarize, the study by Zhang et al. [52] proved that, although the TALENdependent genome transformation strategy has a certain advantage over the CRISPR/Cas9 dependent system, the latter offers significant benefits related to the precision programmed editing of the genes (Table 1). This makes this system a powerful and high-performance genetic engineering tool for livestock breeding practice and, in particular, in the fields of agri-food biotechnology and human food technology (nutritechnology) based on a meat diet [52,68–71].

#### **6. Conclusions and Future Goals**

Although the efficiency of somatic cell cloning in goats remains relatively low, further studies are necessary because modern ART has important implications in the fields of goat breeding, the transgenics of this mammalian species, agri-food biotechnology, biomedicine, and biopharmacy.

An increase in the efficiency of somatic cell cloning techniques in the domestic goat can be brought about by further intensive research into improving both developmental competence and the parameters related to the molecular and epigenetic quality of SCNTderived embryos. The latter can be achieved by efforts aimed at using nonselective or selective inhibitors of DNMTs and HDACs, which would in turn lead to enhancements in the reprogrammability of the epigenetic memory profile within genomic DNA of NDCs, nuclear-transferred oocytes, and the corresponding caprine cloned embryos. This is a sine qua non condition for the practical use of SCNT-based cloning, and thus for the production of genetically transformed goats for the purposes of human nutrition technology based on a meat diet. The main focus of the aforementioned efforts is the successful SCNT-mediated creation and multiplication of transgenic does and bucks with enhanced meat yields due to cellular hyperplasia and hypertrophy within skeletal muscle tissue. This is also a basic requirement for the effective propagation of genetically engineered or genome-edited does for the biopharmaceutical and nutraceutical industry. An ideal example of this is the generation of transgenic goats whose udders serve as bioreactors for recombinant human therapeutic proteins or biochemically humanized milk.

**Author Contributions:** Conceptualization, M.S. (Maria Skrzyszowska) and M.S. (Marcin Samiec); Writing—original draft, M.S. (Maria Skrzyszowska) and M.S. (Marcin Samiec); Writing—review and editing, M.S. (Marcin Samiec); Supervision and funding acquisition, M.S. (Marcin Samiec); Tabular and graphic documentation, M.S. (Marcin Samiec) and M.S. (Maria Skrzyszowska). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Ministry of Education and Science in Poland as a statutory activity No. 04-19-05-00.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

## *Article* **The Relative Abundances of Human Leukocyte Antigen-E,** α**-Galactosidase A and** α**-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells**

**Marcin Samiec 1,\* , Jerzy Wiater 2,\* , Kamil Wartalski <sup>2</sup> , Maria Skrzyszowska <sup>1</sup> , Monika Trzci ´nska <sup>1</sup> , Daniel Lipi ´nski <sup>3</sup> , Jacek Jura <sup>1</sup> , Zdzisław Smor ˛ag <sup>1</sup> , Ryszard Słomski 3,4 and Małgorzata Duda <sup>5</sup>**

**Abstract:** The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from h*FUT2*×h*GLA*×*HLA-E* triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from h*FUT2*, h*GLA* and *HLA-E* transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants. Furthermore, although TSA-based epigenomic modulation has given rise to a remarkable increase in the expression levels of Galα1→3Gal (α-Gal) epitopes that have been determined by lectin blotting analysis, their semi-quantitative profiles have dwindled profoundly in both TSA-exposed and unexposed 3×TG ACFCs as compared to their nTG counterparts. In conclusion, thoroughly exploring proteomic signatures in such epigenetically modulated ex vivo models devised on h*FUT2*×h*GLA*×*HLA-E* triple-transgenic ACFCs that display augmented reprogrammability of translational activities of two mRNA transcripts coding for rhα-Gal A and HLA-E proteins might provide a completely novel and powerful research tool for the panel of further studies. The objective of these future studies should be to multiply the tri-transgenic pigs with the aid of somatic cell nuclear transfer (SCNT)-based cloning for the purposes of both xenografting the porcine cutaneous bioprostheses and dermoplasty-mediated surgical treatments in human patients.

**Keywords:** swine; trichostatin A; epigenetic transformation; ex vivo model; tri-genetically modified; ACFC line; HLA-E; rhα-Gal A; rhα1,2-FT; α-Gal antigenic determinant; porcine skin xenograft

#### **1. Introduction**

At the present stage of investigations in the fields of transplantation medicine and immunology, swine tissues and organs may be an alternative to their human counterparts.

**Citation:** Samiec, M.; Wiater, J.; Wartalski, K.; Skrzyszowska, M.; Trzci ´nska, M.; Lipi ´nski, D.; Jura, J.; Smor ˛ag, Z.; Słomski, R.; Duda, M. The Relative Abundances of Human Leukocyte Antigen-E, α-Galactosidase A and α-Gal Antigenic Determinants Are Biased by Trichostatin A-Dependent Epigenetic Transformation of Triple-Transgenic Pig-Derived Dermal Fibroblast Cells. *Int. J. Mol. Sci.* **2022**, *23*, 10296. https://doi.org/ 10.3390/ijms231810296

Academic Editor: Antonino Germana

Received: 18 August 2022 Accepted: 4 September 2022 Published: 7 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

This is a highlight of research in an era of huge shortages of tissues and organs for allotransplantation, also taking into account a deficiency of allogeneic dermo-epidermal grafts in reconstructive medicine of the human integumentary system and dermoplastybased therapies targeted at cutaneous/subcutaneous tissue engineering. The choice of pigs as donors of xenografts is not accidental. Firstly, anatomohistological and anatomotopographical biocompatibility of porcine tissues, organs and organ systems with their human counterparts determines, to a large extent, closely related physiological sufficiency of organs in these two mammalian species. Secondly, the genetic similarity between humans and domestic pigs is very high, reaching a level oscillating around 96%. Thirdly, *Sus scrofa domesticus* taxon is characterized by tremendously efficient outcomes noticed for species-specific fertility and prolificacy, which makes this livestock species relatively easy to breed [1–5]. Nonetheless, a lack of taxonomic consanguinity occurring between humans and pigs results in the appearance of interspecies immunophysiological incompatibility that largely limits and even prevents completely surgical treatments aimed at xenografting and xenogeneic tissue engineering. This interspecies immunological hindrance is triggered by the presence of a plasmalemma-anchored oligosaccharide moieties of glycoproteins and glycolipids, i.e., Galα(1,3)Galβ(1,4)GlcNAc-R that are designated as the α-Gal antigenic determinants or Galα1→3Gal epitopes on the surface of a vast majority of porcine cells (especially those forming vascular endothelium) [6]. The Galα1→3Gal epitope is common in mammals, but humans and apes have lost this structure through evolution [3,7]. Humans and apes produce natural antibodies against the Galα1→3Gal epitope. Unfortunately, the binding of these antibodies to the Galα1→3Gal epitope leads to hyperacute rejection (HAR) or acute humoral and cellular rejection of porcine xenografts [8–10]. The efforts undertaken to overcome the porcine→human immunological obstacle gave rise to the generation of transgenic pigs exhibiting a reduced or completely abrogated expression of the Galα1→3Gal epitopes [11]. The Galα1→3Gal epitopes can be removed from the surface of porcine cells by stably incorporating the h*FUT2* and h*GLA* gene constructs that is mediated by their intrapronuclear microinjection into porcine fertilized ova (zygotes) [4]. The h*FUT2* gene encodes the enzyme termed as α1,2-fucosyltransferase (H-transferase; α1,2-FT), which is responsible for the formation of the H structure, the core of the system of blood groups AB0 in humans. The action of α1,2-FT blocks the synthesis of the Galα1→3Gal epitope, while promoting the formation of the H structure, which is neutral for the human immune system [12]. The h*GLA* gene encodes human α-galactosidase A (α-Gal A), an enzyme that biocatalyzes the reaction directed to cleave terminal D-galactose residues from the Galα1→3Gal epitope. Deprivation of the Galα1→3Gal epitope of the terminal D-galactose molecules remarkably attenuates its xenoreactivity. This structure becomes undetectable by specific antibodies [13]. Another pivotal problem is the interspecies (porcine→human) immunological incompatibility that arises from phylogenetic divergence and subsequently brings about the species-specific variability at the level of major histocompatibility complex (MHC) proteins. To overcome this limitation, transgenic pigs expressing the *HLA-E* gene are indispensable. The effectiveness of this genetic modification has been proven by research confirming that the presence of human leukocyte antigen-E (HLA-E) molecules in porcine endothelial cells protects them against attack by human NK (natural killer) lymphocytes [14]. Pigs displaying the expression of the *HLA-E* transgene have been successfully created with the aid of intrapronuclear microinjection of the zygotes [15].

It is noteworthy that the applied genetic modification does not always result in a satisfactory (sufficiently high) level of expression of the transgenes integrated with the nuclear host genome. One of the ways to increase the expression of foreign (xenogeneic) genes introduced into the genomic DNA of recipient cells appears to be their epigenetic transformation applying non-specific inhibitors of histone deacetylases (HDACi). Our previous study [16] proved, for the first time, that the use of non-selective HDACi designated as trichostatin A (TSA) for the epigenomic modulation of porcine h*FUT2*×h*GLA* bi-transgenic adult cutaneous fibroblast cells (ACFCs) has contributed to improved reprogrammability followed by increased translational activities identified for mRNA transcripts

synthesized from h*FUT2* and h*GLA* transgenes. Moreover, the action of TSA as a nonspecific epigenetic modifier has been reflected in the augmented expression of pivotal genes coding for structural, enzymatic and other functional proteins indispensable for the biosynthesis and bioaccumulation of the Galα1→3Gal antigenic determinants. However, TSA has been shown to simultaneously enhance the expression of the incorporated h*FUT2*×h*GLA* gene constructs more strongly, so that the extent of Galα1→3Gal epitope silencing estimated for TSA-treated double-transgenic ACFCs was considerably higher than that noticed for TSA-untreated non-transgenic cells. Therefore, the TSA-mediated approach to epigenomically modulate the bi-genetically engineered variant of ACFC lines turned out to be beneficial, since it caused a remarkably diminished incidence of Galα1→3Gal epitopes in this ACFC type [16]. The molecular mechanisms underlying the capabilities of transgenes to epigenetically reprogram their transcriptional activities are complex. They involve amplifying the extent of lysine acetylation (i.e., hyperacetylation) within histones forming chromatin-derived nucleosomal cores. In our previously devised model of bitransgenic ACFCs [16], hyperacetylation seemed to result from alleviation of biocatalytic functions of HDACs by TSA. A TSA-prompted decline in histone deacetylation may also affect other processes, such as the demethylation of DNA cytosine residues, incurring their intensification. A broad spectrum of non-specific HDACi, including TSA, and/or non-specific inhibitors of DNA methyltransferases (DNMTi) and/or selective inhibitors of histone methyltransferases (HMTi) have been formerly utilized in the strategies aimed to epigenomically modulate nuclear recipient oocytes [17–19], nuclear donor cells [20–23] and activated nuclear-transferred oocytes in pigs and other mammalian species [24–26]. These strategies have been developed to predominantly facilitate/refine the reprogramming of epigenomic memory and subsequently enhance the transcriptional activity of donor cell nuclear genomes in mammalian cloned embryos propagated by somatic cell nuclear transfer (SCNT) [27–31].

Taking into consideration all the aforementioned findings, h*FUT2*×h*GLA*×*HLA-E* tri-transgenic ACFC lines also needed to be tested separately by designing their models of the ex vivo migration and expansion under the conditions of TSA-dependent epigenetic transformation. In the current investigation, on the one hand, we have decided to unravel proteomic signatures related to relative abundances (RAs) estimated for HLA-E, rhα-Gal A and rhα1,2-FT in porcine triple-transgenic ACFCs undergoing TSA-assisted epigenomic modulation. On the other hand, the present study sought to decipher semi-quantitative profiles of α-Gal epitopes at the glycoprotein level. This research is the first to comprehensively assess the TSA-expedited enhancement of capabilities of transgene-encoded transcripts to epigenomically reprogram their translational activities in the ex vivo models elaborated to explore proteomic and glycoproteomic profiles in porcine h*FUT2*×h*GLA*×*HLA-E* tri-genetically engineered ACFC lines.

#### **2. Results**

#### *2.1. Western Blot Analysis of the RA Pinpointed for HLA-E, rhα1,2-FT and rhα-Gal A Proteins in the Ex Vivo-Expanded Porcine Triple- and Non-Transgenic ACFCs Undergoing or Not Undergoing TSA-Mediated Epigenetic Transformation*

In vitro proliferating ACFC lines exposed and not exposed to TSA (TSA<sup>+</sup> and TSA−, respectively) were established from dermal explants that had been recovered post-mortem from either h*FUT2*×h*GLA*×*HLA-E* triple-transgenic pigs or their non-transgenic counterparts served as a control group (CTR nTG). Western blot analysis of total protein samples revealed the presence of rhα1,2-FT, rhα-Gal A and HLA-E proteins in all the tri-transgenic samples (Figure 1A). For the TSA− control group, a weak positive signal stemming from all the analyzed proteins was noticed, but it was shown to be unremarkable. In contrast, Western blot analysis of total protein samples derived from TSA<sup>+</sup> ACFCs confirmed the occurrence of clear positive signals for not only rhα1,2-FT but also rhα-Gal A and HLA-E. Signal intensities of analyzed proteins were normalized to β-actin, which was used as a loading control. The semi-quantitative analysis of Western blot strongly supported our findings. Indeed, the relative expression of the HLA-E and rhα-Gal A proteins has been

proven to significantly increase (at least *p* < 0.05) in TSA-treated tri-genetically modified ACFCs as compared to the relevant samples originating from TSA-untreated 3×TG cells (Figure 1B–D). In turn, we did not observe significant differences in the relative expression of rhα1,2-FT between total protein samples derived from TSA<sup>+</sup> and TSA<sup>−</sup> triple-transgenic ACFCs. Interestingly, a clear positive signal was also identified for not only α1,2-FT and α-Gal A enzymes but also swine homolog of the HLA-E (shHLA-E) protein in TSA<sup>+</sup> ACFCs stemming from CTR nTG pigs. This result has been confirmed both qualitatively and semiquantitatively (at least *p* < 0.05) (Figure 1B–D). It is noteworthy that pigs are characterized by the lack of α-Gal A protein expression, which arises from species-specific silencing both alleles of p*GLA* gene. Taking this finding into consideration, the epigenetic alterations resulting from the conditions of either in vitro culture or TSA-assisted epigenomic modulation seem to trigger, to some extent, the onset of transcriptional and translational activities for p*GLA* alleles and their transcribed mRNAs in the ex vivo-expanded porcine CTR nTG ACFCs. TSA-untreated 3×TG cells (Figure 1B–D). In turn, we did not observe significant differences in the relative expression of rhα1,2-FT between total protein samples derived from TSA<sup>+</sup> and TSA<sup>−</sup> triple-transgenic ACFCs. Interestingly, a clear positive signal was also identified for not only α1,2-FT and α-Gal A enzymes but also swine homolog of the HLA-E (shHLA-E) protein in TSA<sup>+</sup> ACFCs stemming from CTR nTG pigs. This result has been confirmed both qualitatively and semi-quantitatively (at least *p* < 0.05) (Figure 1B– D). It is noteworthy that pigs are characterized by the lack of α-Gal A protein expression, which arises from species-specific silencing both alleles of p*GLA* gene. Taking this finding into consideration, the epigenetic alterations resulting from the conditions of either in vitro culture or TSA-assisted epigenomic modulation seem to trigger, to some extent, the onset of transcriptional and translational activities for p*GLA* alleles and their transcribed mRNAs in the ex vivo-expanded porcine CTR nTG ACFCs.

In vitro proliferating ACFC lines exposed and not exposed to TSA (TSA<sup>+</sup> and TSA<sup>−</sup>

respectively) were established from dermal explants that had been recovered postmortem from either h*FUT2×*h*GLA×HLA-E* triple-transgenic pigs or their non-transgenic counterparts served as a control group (CTR nTG). Western blot analysis of total protein samples revealed the presence of rhα1,2-FT, rhα-Gal A and HLA-E proteins in all the tritransgenic samples (Figure 1A). For the TSA<sup>−</sup> control group, a weak positive signal stemming from all the analyzed proteins was noticed, but it was shown to be unremarkable. In contrast, Western blot analysis of total protein samples derived from TSA<sup>+</sup> ACFCs confirmed the occurrence of clear positive signals for not only rhα1,2-FT but also rhα-Gal A and HLA-E. Signal intensities of analyzed proteins were normalized to βactin, which was used as a loading control. The semi-quantitative analysis of Western blot strongly supported our findings. Indeed, the relative expression of the HLA-E and rhα-Gal A proteins has been proven to significantly increase (at least *p* < 0.05) in TSA-treated tri-genetically modified ACFCs as compared to the relevant samples originating from

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*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 19