Pig Cloning Research Articles

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development.

Somatic cell nuclear transfer (SCNT) technology holds great promise for livestock industry, life science and human biomedicine. However, the development and application of this technology is limited by the low developmental potential of SCNT embryos. The developmental competence of cloned embryos is influenced by the energy metabolic status of donor cells. The purpose of this study was to investigate the effects of CPI, an oxidative phosphorylation inhibitor, on the energy metabolism pathways of pig fibroblasts and the development of subsequent SCNT embryos. The results showed that treatment of porcine fibroblasts with CPI changed the cellular energy metabolic pathways from oxidative phosphorylation to glycolysis and enhanced the developmental ability of subsequent SCNT embryos. The present study establishes a simple, new way to improve pig cloning efficiency, helping to promote the development and application of pig SCNT technology.

Identification of Important Factors Causing Developmental Arrest in Cloned Pig Embryos by Embryo Biopsy Combined with Microproteomics.

The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos.

Knockdown of YY1 Inhibits XIST Expression and Enhances Cloned Pig Embryo Development.

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5' regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning.

Enhancement of Chromatin and Epigenetic Reprogramming in Porcine SCNT Embryos-Progresses and Perspectives.

Over the last 25 years, cloned animals have been produced by transferring somatic cell nuclei into enucleated oocytes (SCNT) in more than 20 mammalian species. Among domestic animals, pigs are likely the leading species in the number of clones produced by SCNT. The greater interest in pig cloning has two main reasons, its relevance for food production and as its use as a suitable model in biomedical applications. Recognized progress in animal cloning has been attained over time, but the overall efficiency of SCNT in pigs remains very low, based on the rate of healthy, live born piglets following embryo transfer. Accumulating evidence from studies in mice and other species indicate that new strategies for promoting chromatin and epigenetic reprogramming may represent the beginning of a new era for pig cloning.

TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos.

SummaryPig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.

Application of the modified handmade cloning technique to pigs.

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.

Pig Cloning Using Somatic Cell Nuclear Transfer.

Porcine cloning technology can be used to produce progenies genetically identical to the donor cells from high-quality breeding pigs. In addition, genetically modified pigs have been produced by somatic cell nuclear transfer using genetically modified porcine fetal fibroblasts. The method of preparing genetically modified pigs is critical for establishing pig models for human diseases, and for generating donor animals for future xenotransplantation. This chapter describes detailed procedures for generating cloned pigs using fetal fibroblasts as nuclear donors.

Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear

Cloning, also known as somatic cell nuclear transfer (SCNT), is an asexual reproduction technique that reprograms differentiated cells to the totipotent state, and generates offspring with a genotype identical to the donor cells. Pig cloning technique holds great promise for propagating excellent breeding boars, generating genetically modified pigs, protecting rare and endangered pigs and studying the mechanisms of somatic cell nucleus reprogramming. However, cloned pigs suffer from various developmental defects, including low birth rate, low birth weight, and high stillbirth occurrence, neonatal mortality and congenital malformations, which severely hamper their applications. Errors in epigenetic reprogramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic development in cloned embryos and animals. However, most studies to correct the errors in epigenetic reprogramming of cloned pig embryos have not substantially improved the birth and survival rates of cloned pigs. In this review, we summarize the abnormal phenotypes, causes of abnormal development of cloned pigs and effective methods for improving pig cloning efficiency, thereby providing a reference for the future research to improve the development and survival rates of cloned pig embryos and cloned pigs.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Factors influencing the commercialisation of cloning in the pork industry

Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.

Biotechnological bases of the development of cloned pig embryos

The term ‘clone’ in animal biotechnology refers to an organism derived from non-sexual reproduction, which is both a direct offspring and a genetic copy of the parent organism. To date, the pig appears to be the most interesting object in cloning research. Somatic cell nuclear transfer in pigs has a wide range of potential applications in various fields of human scientific and economic activities. However, the efficiency of producing cloned embryos in swine is still lower than that of other livestock species, in particular horses and cattle. Somatic cell nuclear transfer is a technically complex multi-stage technology, at each stage of which the pig oocytes, which are more susceptible to changes of surrounding conditions, are affected by various factors (mechanical, physical, chemical). At the stage of oocyte maturation, changes in the cell ultrastructures of the ooplasm occur, which play an important role in the subsequent nuclear reprogramming of the transferred donor cell. Before transfer to the oocyte donor somatic cells are synchronized in the G0/G1 stage of the cell cycle to ensure the normal ploidy of the cloned embryo. When removing the nucleus of pig oocytes maturated in vitro, it is necessary to pay attention to the problem of preserving the viability of cells, which were devoid of their own nuclear material. To perform the reconstruction, a somatic cell is placed, using micro-tools, in the perivitelline space, where the first polar body was previously located, or in the cytoplasm of an enucleated oocyte. The method of manual cloning involves the removal of the oocyte nucleus with subsequent fusion with the donor cell without the use of micromanipulation techniques. The increased sensitivity of oocytes to the environmental conditions causes special requirements for the choice of the system for in vitro culture of cloned pig embryos. In this work, we have reviewed the modern methods used for the production of cloned embryos and identified the technological issues that prevent improving the efficiency of somatic cloning of pigs.

Abstract 3706: The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues

Abstract The aim of the project is to generate a toolbox pig model. This is being done because the pig probably is the best non-primate model in translational medicine. Unfortunately, pig models have proven difficult to produce. The two main complications have been the cloning of viable pigs and that just a few genes are altered, which often is not sufficient to induce the desired disease. Especially, models based on multiple genetic modifications of the pig genome, e.g. cancer, are challenging to generate. This was changed with the development of the CRISPR-Cas9 technology, which made it possible to perform multiple in vivo gene editing. We have cloned an inducible Cas9 expressing minipig and have obtained healthy F1 offspring. Activation of the Cas9 expression is attained by transduction with adeno-associated virus that delivers both the recombinase and the guide RNA to the designated tissue. This design provides spatial and temporal control of the activation. The Cas9 expression is mediated by a ubiquitous promotor, which also co-expresses fluorescent proteins for expression analysis. Transgenic Cas9 expression is observed in all major organs with strong expression in the epithelia, muscles and hepatocytes. Successful in vitro gene editing has been conducted in primary keratinocytes and fibroblast obtained from the transgenic pigs, which confirms the genetic design. By delivery of our construct by adeno-associated virus to the skin of the pigs, In vivo activation of the transgenic construct has been achieved. To validate the model, lung cancer has been induced in nine pigs by alteration a panel of cancer related genes including STK11, TP53, PTEN, and KRAS, all key drivers of human lung cancer. To obtain the constitutive active KRAS-G12D isoform of the KRAS oncogene a repair template was supplied. The pigs are being followed by PET/CT scanning in combination with 18F-FDG as a tracer. Preliminary data indicates abnormalities/cancer initiation in the pigs eight months after induction. In conclusion, an inducible Cas9 expressing minipig allows rapid gene editing to study different scenarios of human cancer in a pig model. The development of the Cas9 pig will extend the variety of pig disease models, both to generate a model of lung cancer as proposed but also models for other cancers. Citation Format: Martin F. Berthelsen, Maria Riedel, Mikkel H. Vendelbo, Aage K. Alstrup, Frederik Dagnæs-Hansen, Yonglun Luo, Simone S. Møller, Henrik Callesen, Jannik E. Jakobsen, Martin K. Thomsen. The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3706.

Maternal dietary supplementation of arginine increases the ratio of total cloned piglets born to total transferred cloned embryos by improving the pregnancy rate of recipient sows

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14–75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

SummaryPig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.

Improved Cytotoxic T Lymphocyte Responses to Vaccination with Porcine Reproductive and Respiratory Syndrome Virus in 4-1BB Transgenic Pigs.

Vaccination is the most reliable measure to prevent infectious diseases in domestic animals. Development of novel vaccines demands extensive studies with new technologies, such as using novel adjuvants and immunomodulatory molecules. The co-stimulatory molecule 4-1BB provides a key signal that directs the fate of T cells during activation, and thus is important to their function in immune protection. To determine whether host immune responses to viral infection could be promoted by enhancing 4-1BB co-stimulation, in this study, we produced transgenic pig clones expressing an extra copy of the 4-1BB gene by clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9-mediated homologous recombination at the Rosa26 locus. The immune responses of transgenic pigs to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine were determined on day 14. We show that peripheral blood lymphocytes of transgenic pigs expressed around twice the level of 4-1BB mRNA than those of control pigs. We also found IL-2, TNF-α, and granzyme B mRNA levels as well as PRRSV-specific IFN-γ response were significantly upregulated in 4-1BB transgenic pigs, leading to more efficient cytotoxic T lymphocyte (CTL) killing, whereas the expressions of IL-4, IL-17, and Foxp3 were not affected. These results indicate that higher levels of 4-1BB expression involve in promoting Th1 differentiation and enhancing specific CTL responses to PRRSV, and provide a novel approach to increase the efficacy of current vaccines to control the infectious diseases.

The effect of histone deacetylase inhibitor trichostatin A on porcine mesenchymal stem cell transcriptome

The use of histone deacetylase inhibitors such as trichostatin A (TSA) for epigenetic transformation of mesenchymal stem cells (MSCs), whose nuclei will be transferred into enucleated oocytes, is a novel approach in research involving somatic cell cloning of pigs and other mammalian species. Although the effectiveness of TSA in cloning applications was confirmed, processes and mechanisms underlying achieved effects are not yet fully understood, especially for pig MSCs. To contribute to this knowledge, in this study we performed a comprehensive transcriptome analysis using high-throughput sequencing of pig bone-marrow derived MSCs, both treated and untreated with TSA, and evaluated the effect of TSA administration on their transcription profile after 24 h of in vitro culture. The expression of selected positive and negative mesenchymal surface antigens was also evaluated in these cells by flow cytometry. Subsequently, the stability of induced expression changes was evaluated after another 55–72 h of culture without TSA. The results of this study showed that TSA does not affect the expression of the selected surface antigens related to MSC mesenchymal stemness origin, namely: CD90 (positive marker), CD31 and CD34 (negative markers) and has a wide stimulating effect on MSCs transcription, affecting genes across the whole genome with some minor signs of site-specific acting in regions on SSC2 and SSC6. TSA turned out to have a higher impact on already expressed genes with only minor abilities to induce expression of silenced genes. Genes with expression affected by TSA were related to a wide range of biological processes, however, we found some evidence for specific stimulation of genes associated with development, differentiation, neurogenesis or myogenesis. TSA also seemed to interfere with Wnt signaling pathways by upregulation of several engaged genes. The analysis of cell transcriptome after prolonged culture following the TSA removal, showed that the expression level of majority of genes affected by TSA is restored to the initial level. Nonetheless, the set of about six hundred genes responsible for e.g. adhesion, signal transduction and cell communication was altered even after 55–72 h of culture without TSA. TSA also enhanced expression of some of pluripotency marker genes (FGF2, LIF, TERT) but their expression was stabilized during further culture without TSA. The detailed analysis of factors connected with neuron-like differentiation allowed us to assume that TSA mostly stimulates neurogenic differentiation pathway in the pig MSCs possibly through interaction with Wnt-mediated signaling and thus triggers mechanisms conducive to epigenetic reprograming.

Effects of RNAi-mediated knockdown of Xist on the developmental efficiency of cloned male porcine embryos.

Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.

152 PROLIFERATION- AND SENESCENCE-RELATED GENE EXPRESSION IN CANINE FIBROBLASTS DERIVED FROM DOGS OF DIFFERENT AGE

In pig, cattle and mouse cloning, the age of the cell donor can influence cloning efficiency. However, little is known regarding the effects of the cell donor’s age related to cell proliferation and senescence and its importance for canine cloning. Therefore, the aim of this study was to investigate the effect of cell donor age on growth pattern and doubling time of cell populations, cell size, viability, and senescence-related gene expression (p53, p16, p21, Rb1, and E2F1) in canine fibroblasts. Cells were derived from 1-year-old and 7-year-old dogs. To obtain the fibroblasts, primary culture was performed using ear biopsies from three 1-year-old dogs (1yr) and three 7-year-old dogs (7yr), and cells at passage 2 to 6 were used for somatic cell cloning. Cells were plated at 1 × 105 cells well–1 and harvested every 24 h for 6 days. Cell number was determined for measurement of growth pattern and doubling time. The harvested cells were stained with trypan blue and their size and viability were analysed using a Countess automated cell counter (ThermoFisher Scientific, Waltham, MA, USA). Gene expression was measured by RT-qPCR. Cell proliferation and gene expression experiments were technically replicated 3 and 4 times, respectively. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and t-test (P < 0.05) was used. Growth curves in both groups showed typical S-shape and at Day 6, 1yr samples had significantly higher cell numbers compared with 7yr samples. For doubling times, passages 2 to 5 of 1yr (29.8 ± 4.4 h, 41.0 ± 1.6 h, 49.5 ± 4.8 h, and 71.3 ± 8.4 h) were significantly shorter than those of 7yr (47.0 ± 4.5 h, 57.1 ± 2.3 h, 65.8 ± 1.8 h, and 126.4 ± 12.4 h). Cells from passage 6 of 7yr and passage 7 of 1yr did not reach confluence at Day 6. Cell size of 1yr (14.1 ± 0.2 µm) was significantly smaller than that of 7yr (15.3 ± 0.2 µm). Additionally, there was no significant difference in cell viability between 1yr (90.7 ± 0.8%) and 7yr (88.7 ± 0.7%). However, mRNA expression of p53, p21, and Rb1 was significantly lower in 1yr cells compared with 7yr cells. The expression level of E2F1 was significantly higher in 1yr cells. There was no significant difference between 1yr and 7yr cells for p16 expression. In conclusion, proliferation analysis indicated that somatic cells derived from old donor dogs reach a state of senescence more rapidly compared with those of young donor dogs. In addition, analysis of gene expression demonstrated that cells from old donor dogs have significantly increased p53, p21, and Rb1 expression levels, which indicate senescence. Further studies are needed to produce dogs using 1yr and 7yr cells to evaluate the effect of donor aging in canine somatic cell nuclear transfer efficiency. This study was supported by RAD (#PJ0109282015), IPET (#311062-04-3-SB010), Nature Cell, Research Institute for Veterinary Science, (Natural Balance Korea or Nestle Purina Korea) and the BK21 plus program. We thank Barry D. Bavister for his valuable editing of the manuscript.