Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.

Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.

Extraembryonic Endoderm (XEN) Cells Capable of Contributing to Embryonic Chimeras Established from Pig Embryos.

Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs.

The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.

Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications.

Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3 billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as "dual purpose" applications.

Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene.

Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs.

The domestic pig is an ideal "dual purpose" animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.

Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells.

The development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERß, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.

The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

Ginsenoside Rg1 Improves In vitro-produced Embryo Quality by Increasing Glucose Uptake in Porcine Blastocysts.

Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 (20 µg/mL) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions.

Dosage compensation of X-chromosome inactivation center-linked genes in porcine preimplantation embryos: Non-chromosome-wide initiation of X-chromosome inactivation in blastocysts.

X-chromosome inactivation (XCI) is an epigenetic mechanism that occurs in the eutherian embryo development to equalize the dosage of X-linked genes between males and females. This event is regulated by various factors, and the genes located in the X-chromosome inactivation center (XIC), which is known to be an evolutionary conserved region, are associated with XCI; however, a number of studies regarding this epigenetic event and genomic region are primarily performed in mouse models despite its species-specific features. Thus, in this study, the porcine XIC was identified, and we analyzed the expression of XIC-linked genes in porcine preimplantation embryos. Comparative sequence analysis revealed that the porcine XIC is synteny with that of human and the non-coding RNAs were less conserved compared with the protein coding genes in the XIC. Among the XIC-linked genes, the expression levels of CHIC1 and RLIM were decreased from morula to blastocyst development and their dosage was compensated between the male and female blastocysts. Additionally, the CpG sites of CHIC1 were approximately 50% methylated in parthenote blastocysts. Contrary to these genes, XIST and LOC102165544, an uncharacterized non-coding gene, showed dramatically increased expression levels after the morula stage and preferential female expression in blastocysts. Imprinted XIST expression was not observed, and their CpG sites were hypo-methylated in parthenogenic blastocysts. These results demonstrate that the porcine XIC consists of an evolutionary conserved structure with fewer sequences conserved non-coding RNAs. In addition, a few XIC-linked genes would likely achieve dosage compensation, but XCI would not be completed in porcine blastocysts.

Identification and differential expression patterns of porcine OCT4 variants.

OCT4 encoded by POU5F1 has a crucial role of maintaining pluripotency in embryonic stem cells during early embryonic development and several OCT4 variants have been identified in mouse and human studies. The objective of this study was to identify different variants of OCT4 and analyze their expression patterns in preimplantation porcine embryos and various tissues. In this study, we showed that POU5F1 transcribes its three variants, namely OCT4A, OCT4B, and OCT4B1. The OCT4B transcript consists of exons identical to the major form of the OCT4 variant, OCT4A, with a differential N-terminal domain-coding exon. The structure of OCT4B1 mRNA was the same as that of OCT4B mRNA, but harbored a cryptic exon. Based on these findings, the transcription levels were investigated and found that OCT4B and OCT4B1 made up ∼20% among the variants in the embryonic stage and this indicates that OCT4A mRNA is dominantly expressed during preimplantation embryo development. In addition, OCT4B mRNA was detected in all tissues examined, while OCT4A and OCT4B1 were detected only in testis but not in other tissues examined. OCT4B1 showed inversely correlated expression with SOX2 and NANOG expression. OCT4A protein was specifically localized to the nuclei, whereas OCT4B was mainly localized to the cytoplasm of the porcine embryos at the blastocyst stage. The findings of this study reveal that the porcine OCT4 gene can potentially encode three variants (OCT4A, OCT4B, and OCT4B1), and they are differentially expressed and would have roles dissimilar between each other in preimplantation embryos and various adult tissues.

Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos.

Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs.

To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVF- and three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general "stemness" criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development.

Availability of empty zona pellucida for generating embryonic chimeras.

In the present study we used an empty zona pellucida derived from hatched blastocysts as an alternative source for embryo aggregation and compared results with the conventional microwell method. Denuded 4-cell stage porcine embryos were aggregated by introduction into an empty zona or placement within a concave microwell. The present study showed that although the rate of aggregate formation was similar, the blastocyst rates and allocation of more cells to the inner cell mass (ICM) in the resultant aggregates were increased significantly more in the empty zona than in the microwell. Notably, using an empty zona showed no limitations with regards to the increased number of embryos aggregated or embryonic stages for aggregation, while partial or no aggregation frequently occurred in the microwell. The discrepancy may be due to the difference of microenvironments where the embryos were placed namely, the presence/absence of zona pellucida. We hypothesize the success of the empty zona in generating aggregates is due to the physical aggregation of individual embryos allowing closer contact between the blastomeres and/or embryos compared with a concave microwell. These results indicate that aggregation conditions could influence overall production efficiency and developmental potential of aggregates, suggesting physical restraint via empty zona that provide three-dimensional pressures is an important factor for successful embryo aggregation.

Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.

Effects of Trichostatin A on In vitro Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer.

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos.

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.