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

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (30 September 2022) | Viewed by 38906

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Prof. Dr. Marcin Samiec

E-Mail Website
Guest Editor
Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Balice near Kraków, 32-083 Balice, Poland
Interests: reproductive biology and biotechnology in different mammalian species (especially pigs, goats, cattle, rabbits and horses); assisted reproductive technologies (ARTs)—experimental and applied embryology; intra- and interspecies somatic cell cloning by somatic cell nuclear transfer (SCNT); transgenic research; parthenogenetic activation of oocytes (methods and molecular mechanisms of oocyte activation); in vitro embryo production (IVP)—in vitro oocyte maturation (IVM), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), in vitro embryo culture (IVC); stem cell research; 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; unravelling the transcriptomic and proteomic profiles in nuclear donor cell lines (somatic and stem cell lines) and nuclear recipient oocytes; the ART-mediated programs focused on biotechnological, transgenic and biomedical research
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Maria Skrzyszowska

E-Mail Website
Guest Editor
Department of Reproductive Biotechnology and Cryoconservation, National Research Institute of Animal Production, Balice near Kraków, 32-083 Balice, Poland
Interests: animal reproduction biotechnology; somatic cell cloning in different mammalian species; embryonic cell cloning in different mammalian species, including embryo bisection methods; in vitro embryo production; in vitro oocyte maturation and microsurgical in vitro fertilization by ICSI; transgenesis; modern strategies to assess the molecular quality of somatic cell lines and female gametes for the purposes of advanced ARTs
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Although approximately 25 mammalian species have been cloned by somatic cell nuclear transfer (SCNT) so far, the efficiency of somatic cell cloning is still at a disappointingly low level ranging from 0.1% to less than 5% as measured by the percentage of the cloned progeny generated to the number of oocytes reconstructed by SCNT. Therefore, the studies focused on somatic cell cloning require an important milestone to improve the pre- and/or post-implantation capabilities and enhance the molecular quality of cloned mammalian embryos. This finding seems to be a sine qua non condition for increasing the effectiveness of SCNT as assisted reproductive technology (ART) and its potential of wide application to embryological, biotechnological, transgenic and biomedical research.

To sum up, this Special Issue offers/opens the possibility of publishing the articles and reviews aimed at exploring or recognizing a variety of molecular and epigenetic determinants of the SCNT efficacy at the levels of nuclear donor cells, nuclear recipient oocytes and nuclear-transferred embryos. Their broad identification can contribute to amelioration, optimization and implementation of the somatic cell cloning. These latter allow this method to become one of the most important, reliable and feasible ART strategies for the purposes of experimental and applied embryology, biotechnology, ex situ biodiversity preservation, transgenics, biomedicine, biopharmacy and creation of animal models for etiopathogenesis and pathophysiology of human diseases.

Prof. Dr. Marcin Samiec
Prof. Dr. Maria Skrzyszowska
Guest Editors

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Keywords

  • somatic cell nuclear transfer (SCNT)
  • assisted reproductive technology (ART)
  • somatic cell cloning

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Published Papers (11 papers)

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Editorial

Jump to: Research, Review

5 pages, 235 KiB  
Editorial
Molecular Mechanism and Application of Somatic Cell Cloning in Mammals—Past, Present and Future
by Marcin Samiec
Int. J. Mol. Sci. 2022, 23(22), 13786; https://doi.org/10.3390/ijms232213786 - 9 Nov 2022
Cited by 9 | Viewed by 2283
Abstract
Thus far, nearly 25 mammalian species have been cloned by intra- or interspecies somatic cell nuclear transfer (SCNT) [...] Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)

Research

Jump to: Editorial, Review

15 pages, 1599 KiB  
Article
Trichostatin A-Mediated Epigenetic Modulation Predominantly Triggers Transcriptomic Alterations in the Ex Vivo Expanded Equine Chondrocytes
by Tomasz Ząbek, Wojciech Witarski, Tomasz Szmatoła, Sebastian Sawicki, Justyna Mrozowicz and Marcin Samiec
Int. J. Mol. Sci. 2022, 23(21), 13168; https://doi.org/10.3390/ijms232113168 - 29 Oct 2022
Viewed by 1729
Abstract
Epigenetic mechanisms of gene regulation are important for the proper differentiation of cells used for therapeutic and regenerative purposes. The primary goal of the present study was to investigate the impacts of 5-aza-2′ deoxycytidine (5-AZA-dc)- and/or trichostatin A (TSA)-mediated approaches applied to epigenomically [...] Read more.
Epigenetic mechanisms of gene regulation are important for the proper differentiation of cells used for therapeutic and regenerative purposes. The primary goal of the present study was to investigate the impacts of 5-aza-2′ deoxycytidine (5-AZA-dc)- and/or trichostatin A (TSA)-mediated approaches applied to epigenomically modulate the ex vivo expanded equine chondrocytes maintained in monolayer culture on the status of chondrogenic cytodifferentiation at the transcriptome level. The results of next-generation sequencing of 3′ mRNA-seq libraries on stimulated and unstimulated chondrocytes of the third passage showed no significant influence of 5-AZA-dc treatment. Chondrocytes stimulated with TSA or with a combination of 5-AZA-dc+TSA revealed significant expressional decline, mainly for genes encoding histone and DNA methyltransferases, but also for other genes, many of which are enriched in canonical pathways that are important for chondrocyte biology. The TSA- or 5-AZA-dc+TSA-induced upregulation of expanded chondrocytes included genes that are involved in histone hyperacetylation and also genes relevant to rheumatoid arthritis and inflammation. Chondrocyte stimulation experiments including a TSA modifier also led to the unexpected expression incrementation of genes encoding HDAC3, SIRT2, and SIRT5 histone deacetylases and the MBD1 CpG-binding domain protein, pointing to another function of the TSA agent besides its epigenetic-like properties. Based on the transcriptomic data, TSA stimulation seems to be undesirable for chondrogenic differentiation of passaged cartilaginous cells in a monolayer culture. Nonetheless, obtained transcriptomic results of TSA-dependent epigenomic modification of the ex vivo expanded equine chondrocytes provide a new source of data important for the potential application of epigenetically altered cells for transplantation purposes in tissue engineering of the equine skeletal system. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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19 pages, 2162 KiB  
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
by Marcin Samiec, Jerzy Wiater, Kamil Wartalski, Maria Skrzyszowska, Monika Trzcińska, Daniel Lipiński, Jacek Jura, Zdzisław Smorąg, Ryszard Słomski and Małgorzata Duda
Int. J. Mol. Sci. 2022, 23(18), 10296; https://doi.org/10.3390/ijms231810296 - 7 Sep 2022
Cited by 14 | Viewed by 2070
Abstract
The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome [...] Read more.
The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×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 hFUT2, hGLA 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 hFUT2×hGLA×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. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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16 pages, 2556 KiB  
Article
Tracking the Molecular Scenarios for Tumorigenic Remodeling of Extracellular Matrix Based on Gene Expression Profiling in Equine Skin Neoplasia Models
by Przemysław Podstawski, Katarzyna Ropka-Molik, Ewelina Semik-Gurgul, Marcin Samiec, Maria Skrzyszowska, Zenon Podstawski, Tomasz Szmatoła, Maciej Witkowski and Klaudia Pawlina-Tyszko
Int. J. Mol. Sci. 2022, 23(12), 6506; https://doi.org/10.3390/ijms23126506 - 10 Jun 2022
Cited by 2 | Viewed by 2494
Abstract
An important component of tissues is the extracellular matrix (ECM), which not only forms a tissue scaffold, but also provides the environment for numerous biochemical reactions. Its composition is strictly regulated, and any irregularities can result in the development of many diseases, including [...] Read more.
An important component of tissues is the extracellular matrix (ECM), which not only forms a tissue scaffold, but also provides the environment for numerous biochemical reactions. Its composition is strictly regulated, and any irregularities can result in the development of many diseases, including cancer. Sarcoid is the most common skin cancer in equids. Its formation results from the presence of the genetic material of the bovine papillomavirus (BPV). In addition, it is assumed that sarcoid-dependent oncogenic transformation arises from a disturbed wound healing process, which may be due to the incorrect functioning of the ECM. Moreover, sarcoid is characterized by a failure to metastasize. Therefore, in this study we decided to investigate the differences in the expression profiles of genes related not only to ECM remodeling, but also to the cell adhesion pathway, in order to estimate the influence of disturbances within the ECM on the sarcoid formation process. Furthermore, we conducted comparative research not only between equine sarcoid tissue bioptates and healthy skin-derived explants, but also between dermal fibroblast cell lines transfected and non-transfected with a construct encoding the E4 protein of the BP virus, in order to determine its effect on ECM disorders. The obtained results strongly support the hypothesis that ECM-related genes are correlated with sarcoid formation. The deregulated expression of selected genes was shown in both equine sarcoid tissue bioptates and adult cutaneous fibroblast cell (ACFC) lines neoplastically transformed by nucleofection with gene constructs encoding BPV1-E1^E4 protein. The identified genes (CD99, ITGB1, JAM3 and CADM1) were up- or down-regulated, which pinpointed the phenotypic differences from the backgrounds noticed for adequate expression profiles in other cancerous or noncancerous tumors as reported in the available literature data. Unravelling the molecular pathways of ECM remodeling and cell adhesion in the in vivo and ex vivo models of epidermal/dermal sarcoid-related cancerogenesis might provide powerful tools for further investigations of genetic and epigenetic biomarkers for both silencing and re-initiating the processes of sarcoid-dependent neoplasia. Recognizing those biomarkers might insightfully explain the relatively high capacity of sarcoid-descended cancerous cell derivatives to epigenomically reprogram their nonmalignant neoplastic status in domestic horse cloned embryos produced by somatic cell nuclear transfer (SCNT). Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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36 pages, 4562 KiB  
Article
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
by Gabriela Gorczyca, Kamil Wartalski, Marek Romek, Marcin Samiec and Małgorzata Duda
Int. J. Mol. Sci. 2022, 23(9), 4572; https://doi.org/10.3390/ijms23094572 - 20 Apr 2022
Cited by 28 | Viewed by 3658
Abstract
Thus far, the potential short- and long-term detrimental effects of a variety of environmental chemicals designated as endocrine-active compounds (EACs) have been found to interfere with histo- and anatomo-physiological functions of the reproductive system in humans and wildlife species. For those reasons, this [...] Read more.
Thus far, the potential short- and long-term detrimental effects of a variety of environmental chemicals designated as endocrine-active compounds (EACs) have been found to interfere with histo- and anatomo-physiological functions of the reproductive system in humans and wildlife species. For those reasons, this study sought to examine whether selected EACs, which encompass the fungicide vinclozolin (Vnz), the androgenic anabolic steroid nandrolone (Ndn) and the immunosuppressant cyclosporin A (CsA), affect the developmental competence and molecular quality (MQ) of porcine cumulus–oocyte complexes (COCs) subjected to in vitro maturation (IVM) under 3D culture conditions. The COCs underwent 3D-IVM in the presence of Vnz, Ndn or CsA for 48 h. To explore whether the selected EACs induce internucleosomal DNA fragmentation in cumulus cells (CCs), TUNEL-assisted detection of late apoptotic cells was performed. Additionally, for the detailed evaluation of pro- and antiapoptotic pathways in COCs, apoptosis proteome profiler arrays were used. To determine changes in intracellular metabolism in COCs, comprehensive assessments of mitochondrial ultrastructure and activity were carried out. Moreover, the relative abundances (RAs) of mRNAs transcribed from genes that are involved in scavenging reactive oxygen species (ROS), such as SIRT3 and FOXO3, and intramitochondrial bioenergetic balance, such as ATP synthase subunit (ATP5A1), were ascertained. Finally, to investigate the extent of progression of oocyte maturation, the intraooplasmic levels of cAMP and the RAs of mRNA transcripts encoding regulatory and biocatalytic subunits of a heterodimeric meiosis-promoting factor, termed cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2), were also estimated. The obtained results provide, for the first time, strong evidence that both Vnz and Ndn decrease the developmental competence of oocytes and stimulate apoptosis processes in CCs. The present study is also the first to highlight that Vnz accelerates the maturation process in immature oocytes due to both increased ROS production and the augmented RA of the CCNB1 gene. Furthermore, Vnz was proven to trigger proapoptotic events in CCs by prompting the activity of the FOXO3 transcription factor, which regulates the mitochondrial apoptosis pathway. In turn, Ndn was shown to inhibit oocyte maturation by inducing molecular events that ultimately lead to an increase in the intraooplasmic cAMP concentration. However, due to the simultaneous enhancement of the expression of TNF-β and HSP27 proteins in CCs, Ndn might be responsible for the onset of their neoplastic transformation. Finally, our current investigation is the first to clearly demonstrate that although CsA did not interfere with the nuclear and cytoplasmic maturation of oocytes, by inducing mitophagy in CCs, it disrupted oocyte metabolism, consequently attenuating the parameters related to the MQ of COCs. Summing up, Vnz, Ndn and CsA reduced not only the processes of growth and IVM but also the MQ of porcine COCs, which might make them unsuitable for assisted reproductive technologies (ARTs) such as in vitro fertilization by either gamete co-incubation or intracytoplasmic sperm injection (ICSI) and cloning by somatic cell nuclear transfer (SCNT). Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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24 pages, 3764 KiB  
Article
The Induced Expression of BPV E4 Gene in Equine Adult Dermal Fibroblast Cells as a Potential Model of Skin Sarcoid-like Neoplasia
by Przemysław Podstawski, Marcin Samiec, Maria Skrzyszowska, Tomasz Szmatoła, Ewelina Semik-Gurgul and Katarzyna Ropka-Molik
Int. J. Mol. Sci. 2022, 23(4), 1970; https://doi.org/10.3390/ijms23041970 - 10 Feb 2022
Cited by 6 | Viewed by 2448
Abstract
The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult [...] Read more.
The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that BPV-E4- and BPV-E1^E4-mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like neoplastic transformation of equine ACFCs. Furthermore, the results of the current investigation might contribute to the creation of in vitro biomedical models suitable for estimating the fates of molecular dedifferentiability and the epigenomic reprogrammability of BPV-E4 and BPV-E4^E1 transgenic equine ACFC-derived sarcoid-like cell nuclei in equine somatic cell-cloned embryos. Additionally, these in vitro models seem to be reliable for thoroughly recognizing molecular mechanisms that underlie not only oncogenic alterations in transcriptomic signatures, but also the etiopathogenesis of epidermal and dermal sarcoid-dependent neoplastic transformations in horses and other equids. For those reasons, the aforementioned transgenic models might be useful for devising clinical treatments in horses afflicted with sarcoid-related neoplasia of cutaneous and subcutaneous tissues. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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30 pages, 3436 KiB  
Article
Brachygnathia Inferior in Cloned Dogs Is Possibly Correlated with Variants of Wnt Signaling Pathway Initiators
by Yong-ho Choe, Tai-Young Hur, Sung-Lim Lee, Seunghoon Lee, Dajeong Lim, Bong-Hwan Choi, Haeyun Jeong, Jin-Gu No and Sun A Ock
Int. J. Mol. Sci. 2022, 23(1), 475; https://doi.org/10.3390/ijms23010475 - 1 Jan 2022
Cited by 4 | Viewed by 2427
Abstract
Abnormalities in animals cloned via somatic cell nuclear transfer (SCNT) have been reported. In this study, to produce bomb-sniffing dogs, we successfully cloned four healthy dogs through SCNT using the same donor genome from the skin of a male German shepherd old dog. [...] Read more.
Abnormalities in animals cloned via somatic cell nuclear transfer (SCNT) have been reported. In this study, to produce bomb-sniffing dogs, we successfully cloned four healthy dogs through SCNT using the same donor genome from the skin of a male German shepherd old dog. Veterinary diagnosis (X-ray/3D-CT imaging) revealed that two cloned dogs showed normal phenotypes, whereas the others showed abnormal shortening of the mandible (brachygnathia inferior) at 1 month after birth, even though they were cloned under the same conditions except for the oocyte source. Therefore, we aimed to determine the genetic cause of brachygnathia inferior in these cloned dogs. To determine the genetic defects related to brachygnathia inferior, we performed karyotyping and whole-genome sequencing (WGS) for identifying small genetic alterations in the genome, such as single-nucleotide variations or frameshifts. There were no chromosomal numerical abnormalities in all cloned dogs. However, WGS analysis revealed variants of Wnt signaling pathway initiators (WNT5B, DVL2, DACT1, ARRB2, FZD 4/8) and cadherin (CDH11, CDH1like) in cloned dogs with brachygnathia inferior. In conclusion, this study proposes that brachygnathia inferior in cloned dogs may be associated with variants in initiators and/or regulators of the Wnt/cadherin signaling pathway. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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23 pages, 2508 KiB  
Article
Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation
by Gabriela Gorczyca, Kamil Wartalski, Jerzy Wiater, Marcin Samiec, Zbigniew Tabarowski and Małgorzata Duda
Int. J. Mol. Sci. 2021, 22(21), 11800; https://doi.org/10.3390/ijms222111800 - 30 Oct 2021
Cited by 23 | Viewed by 2877
Abstract
Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn [...] Read more.
Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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17 pages, 3603 KiB  
Article
Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies
by Jerzy Wiater, Marcin Samiec, Kamil Wartalski, Zdzisław Smorąg, Jacek Jura, Ryszard Słomski, Maria Skrzyszowska and Marek Romek
Int. J. Mol. Sci. 2021, 22(18), 9683; https://doi.org/10.3390/ijms22189683 - 7 Sep 2021
Cited by 30 | Viewed by 2984
Abstract
Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and [...] Read more.
Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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Review

Jump to: Editorial, Research

15 pages, 680 KiB  
Review
Generating Cloned Goats by Somatic Cell Nuclear Transfer—Molecular Determinants and Application to Transgenics and Biomedicine
by Maria Skrzyszowska and Marcin Samiec
Int. J. Mol. Sci. 2021, 22(14), 7490; https://doi.org/10.3390/ijms22147490 - 13 Jul 2021
Cited by 32 | Viewed by 8262
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 [...] Read more.
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 genome-edited 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 SCNT-derived goats for the purposes of pharmacology, biomedicine, nutritional biotechnology, the agri-food industry, and modern livestock breeding. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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25 pages, 1506 KiB  
Review
Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals
by Marcin Samiec and Maria Skrzyszowska
Int. J. Mol. Sci. 2021, 22(6), 3099; https://doi.org/10.3390/ijms22063099 - 18 Mar 2021
Cited by 35 | Viewed by 6096
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Molecular Mechanism and Application of Somatic Cell Cloning in Mammals)
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