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.

Comprehensive characteristics of microRNA expression profile of equine sarcoids.

Equine sarcoids are the most common neoplasms occurring in horses. Despite frequent occurrence, they are still not well described at the molecular level. Thus, in the present study, we performed a comprehensive comparative analysis of sarcoid miRNAome profile to identify aberrantly expressed microRNAs, along with their structural variants, potentially useful as biomarkers and, in a wider perspective, broaden the knowledge about this tumor and underlying mechanisms. To this end, we conducted next generation sequencing and as a result we identified both known and potentially novel miRNAs. Differential expression analysis revealed the existence of almost one hundred miRNAs being over- or underexpressed in sarcoids in comparison to healthy tissue (p-adj<0.05), of which many are known for their involvement in processes crucial for neoplastic transformation. Among upregulated miRNAs there were those associated with decreased cell adhesion abilities as well as engaged in global protein production, while downregulation of some miRs i.a. increased cell expansion abilities. Moreover, we identified altered expression levels of miRNA variants (isomiRs) between the investigated tissues. Further analysis revealed that 5' isomiRs comprise different seed sequences leading to target gene switching followed by activation of different biological pathways. Our results are the first which revealed the complexity of microRNA profiles in equine sarcoids and skin tissue, along with the dynamism of their growing in importance concomitants, namely isomiRs. They also showed miRNA molecules and biological pathways important from the sarcoid oncogenesis point of view.

The characteristics of the porcine (Sus scrofa) liver miRNAome with the use of next generation sequencing.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs, which play a vital role in the regulation of gene expression by binding to the 3' untranslated region (3'UTR) of a target mRNA. Despite a significant improvement in the identification of miRNAs in a variety of species, the coverage of the porcine miRNAome is still scarce. To identify porcine miRNAs potentially regulating processes taking place in the liver, we applied next generation sequencing. As a result, we detected 206 distinct miRNAs, of which 68 represented potential novel miRNAs. Among these new miRNAs, there were miRNAs deriving from the opposite arm of a hairpin precursor of already known miRNAs. Moreover, we observed 3' and 5' length and sequence variants, probably constituting so called isomiRs, as well as differentially mapped precursor loci, alternative precursor sequences and clustering of miRNA encoding genes. On the basis of expression levels, reflected by the number of sequence reads, we identified the most abundant miRNAs followed by gene target prediction and pathway analysis. The enriched pathways were connected with cellular and metabolic processes, growth factors as well as enzymatic activity. The obtained results are the first ones to concern the porcine liver miRNAome. Consequently, they will increase the number of known porcine miRNAs and facilitate further research on gene regulation mechanisms as well as biological processes associated with the liver functioning in pigs.

Identification of differential selection traces in two Polish cattle breeds.

Genetic improvement of animals based on artificial selection is leading to changes in the frequency of genes related to desirable production traits. The changes are reflected by the neutral, intergenic single nucleotide polymorphims (SNPs) being in long-range linkage disequilibrium with functional polymorphisms. Genome-wide SNP analysis tools designed for cattle, allow for scanning divergences in allelic frequencies between distinct breeds and thus for identification of genomic regions which were divergently selected in breeds' histories. In this study, by using Bovine SNP50 assay, we attempted to identify genomic regions showing the highest differences in allele frequencies between two distinct cattle breeds - preserved, unselected Polish Red breed and highly selected Holstein cattle. Our study revealed 19 genomic regions encompassing 55 protein-coding genes and numerous quantitative trait loci which potentially may underlie some of the phenotypic traits distinguishing the breeds.

Sex reversal syndrome in the horse: four new cases of feminization in individuals carrying a 64,XY SRY negative chromosomal complement.

Horses are characterized as having a greater rate of chromosomal abnormalities than other species, which are mainly related to the sex chromosome pair and produce a series of different anomalies known as disorders in sexual development (DSD). In the present study, three Pura Raza Española (PRE) and one Menorquín (MEN) horses were studied and an incompatibility in their genetic and phenotypic sex were detected. Animals were karyotyped by conventional and molecular cytogenetic analyses and characterized using genomic techniques. Although all individuals, were totally unrelated, these animals had the same abnormality (64,XY SRY negative DSD) despite having an anatomically normal external mare phenotype. Therefore, this syndrome could remain undiagnosed in a large percentage of cases because the physiological and morphological symptoms are rare. In the present study, a slight gonadal dysgenesis was observed only in older individuals. Interestingly this chromosomal abnormality has been previously reported less than twenty times, and never in the PRE or MEN horses. With the present research, it is demonstrated that the use of genetic and cytogenetic diagnostic tools in veterinary practice could be an important complementary test to determine the origin of unexplained reproductive failures among horses.

Gene mapping as a method for verifying sequence localization based on interspecific chromosome painting (ZOO-FISH).

The results obtained in the present study made it possible to place selected markers on the physical map of the arctic fox genome. With the use of fluorescence in situ hybridization (FISH) the GHR (3q24) and 1110 (1q21.1-21.2) genes and the FH2537 (5q11.3) microsatellite were localized on arctic fox chromosomes. The results confirmed previously proposed homologies using the ZOO-FISH technique, except for the 1110 gene. This suggests that the gene underwent a rearrangement (an inversion) that changed its localization compared to the dog.

The application of genome-wide SNP genotyping methods in studies on livestock genomes.

Animal genomics is currently undergoing dynamic development, which is driven by the flourishing of high-throughput genome analysis methods. Recently, a large number of animals has been genotyped with the use of whole-genome genotyping assays in the course of genomic selection programmes. The results of such genotyping can also be used for studies on different aspects of livestock genome functioning and diversity. In this article, we review the recent literature concentrating on various aspects of animal genomics, including studies on linkage disequilibrium, runs of homozygosity, selection signatures, copy number variation and genetic differentiation of animal populations. Our work is aimed at providing insight into certain achievements of animal genomics and to arouse interest in basic research on the complexity and structure of the genomes of livestock.

General assessment of copy number variation in normal and tumor tissues of the domestic dog (Canis lupus familiaris).

In recent years, characterization of a copy number variation (CNV) of the genomic DNA has provided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. Copy number variants (CNVs) also occur in the genomes of healthy individuals as a result of abnormal recombination processes in germ cells and have a hereditary character contributing to the natural genetic diversity. Recent image analysis methods and advanced computational techniques allow for identification of CNVs using SNPs genotyping microarrays based on the analysis of signal intensity observed for markers located in the specific genomic regions. In this study we used CanineHD BeadChip assay (Illumina) to identify both natural and cancer-induced CNVs in the genomes of different dog breeds and in different cancer types occurring in this species. The obtained results showed that structural aberrations are a common phenomenon arising during a tumor progression and are more complex and widespread in tumors of mesenchymal tissue origin than in epithelial tissue originating tumors. The tumor derived CNVs, in comparison to healthy samples, were characterized by larger sizes of regions, higher number of amplifications, and in some cases encompassed genes with potential effect on tumor progression.

Analysis of genomic instability in primary spermatocytes of interspecific hybrids of the red fox (Vulpes vulpes) and the Arctic fox (Alopex lagopus).

The aim of this study was to analyse meiotic cells of male interspecific hybrids of the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus). To this end we determined stages of meiotic cells as well as carried out FISH analyses with probes specific to heterosomes and a TUNEL assay on synaptonemal complex preparations. The meiotic cell analysis revealed only the presence of stages of the first meiotic division from leptotene to pachytene. Moreover, we observed an increased level of early dissociation of the X-Y bivalent as well as a high percentage of apoptotic cells. These results indicate the disruption of meiotic division in male hybrids manifested through meiotic arrest of the cells. Faulty pairing of the heterosomes can be considered as one of the causes leading to the initiation of the apoptotic process.

The use of primed in situ synthesis (PRINS) to analyze nucleolar organizer regions (NORs) and telomeric DNA sequences in the domestic chicken genome.

One of the most often analyzed avian genomes is the domestic chicken genome (Gallus domesticus) whose diploid number is 2n = 78. In the chicken karyotype, similarly to other birds, there is a group of microchromosomes for which the determination of morphology and banding pattern is impossible using classic cytogenetics methods. The aim of this study was to evaluate telomeric and rDNA repetitive sequences in the chicken genome by the PRINS technique as an alternative method to fluorescence in situ hybridization. This is the first report on the application of the PRINS method to locate these repetitive sequences in the chicken nuclei and metaphase chromosomes.

Polymorphism of cytogenetic markers in wild and farm red fox (Vulpes vulpes) populations.

Analysis of the origin of domestic animals is of wide interest and has many practical applications in areas such as agriculture and evolutionary biology. Identification of an ancestor and comparison with the domesticated form allows for an analysis of genetic, physiological, morphological and behavioral effects of domestication. Because fox breeding has been an ongoing process for over a century, differences are expected between farm and wild populations at the chromosomal level. The aim of this work was to analyse polymorphisms at the chromosomal level in foxes raised on farms and those living in the wild. Blood samples and lung tissue served as the experimental material and were obtained after slaughter of 35 foxes, including 28 breeding animals and 7 wild animals. The classical cytogenetic method was used including AgNOR technique, as well as molecular methods such as fluorescence in situ hybridization (FISH), and primed in situ labeling (PRINS). Analysis of the number of B chromosomes showed the presence of polymorphisms in foxes from both studied populations, but there was no correlation between the number of B chromosomes and the origin and gender of particular animals. An analysis ofactive nucleolar organizers showed the presence of a large number of polymorphisms and a tendency towards reduction of the number of NORs in the captive-raised population.

The evaluation of bovine SNP50 beadchip assay performance in Polish red cattle breed.

According to increasing interest in the use of high density SNP (single nucleotide polymorphism) genotyping assays for genome-wide genetic studies in farm animals, there is a need to assess the usefulness of currently available genomic tools for application in different breeds. The performance of the assays may differ between the breeds because of discrepancies in allele frequencies of the polymorphisms or differences in linkage disequilibrium patterns. In this study we attempted to test the performance of the Bovine SNP50 v2 genotyping assay (Illumina) for population genetic and other applications in the Polish Red cattle breed. We found that 37,977 of the 53,438 autosomal markers included in the assay give high quality genomic information and can be used for different applications concerning this breed. The remaining markers were denoted as "of limited use" or redundant because of their weak performance, deviation from Hardy-Weinberg equilibrium, low minor allele frequency, high linkage disequilibrium with other neighboring markers or location on sex chromosomes.

The evaluation of the usefulness of pedigree verification-dedicated SNPs for breed assignment in three polish cattle populations.

The breed assignment in cattle is one of the issues of molecular genetics which needs further testing and development. Although several statistical approaches have been developed to enable such application, the obtained results strongly depend on specific populations differentiation and power of markers discrimination or their informativeness. Currently, all breeding animals are being tested for parentage with the use of panel of 12 microsatellite markers, which in near future probably will be replaced by about 100 single nucleotide polymorphisms (SNPs). Despite the fact that SNPs are mainly bi-allelic, the multilocus genotypes can reach the level of polymorphism of a panel of microsatellite markers. In this study we attempted to determine the breed of origin of 741 cattle by using 120 SNPs dedicated for parentage testing and included in the BovineSNP50 BeadChip genotyping assay (Illumina). The applied Bayesian and frequency-based methods allowed such differentiation, however, the reliability of the results was not completely satisfying, suggesting that the studied markers are not the best tool for breed assignment.

Comparative cytogenetic analysis of sex chromosomes in several Canidae species using zoo-FISH.

Sex chromosome differentiation began early during mammalian evolution. The karyotype of almost all placental mammals living today includes a pair of heterosomes: XX in females and XY in males. The genomes of different species may contain homologous synteny blocks indicating that they share a common ancestry. One of the tools used for their identification is the Zoo-FISH technique. The aim of the study was to determine whether sex chromosomes of some members of the Canidae family (the domestic dog, the red fox, the arctic fox, an interspecific hybrid: arctic fox x red fox and the Chinese raccoon dog) are evolutionarily conservative. Comparative cytogenetic analysis by Zoo-FISH using painting probes specific to domestic dog heterosomes was performed. The results show the presence of homologous synteny covering the entire structures of the X and the Y chromosomes. This suggests that sex chromosomes are conserved in the Canidae family. The data obtained through Zoo-FISH karyotype analysis append information obtained using other comparative genomics methods, giving a more complete depiction of genome evolution.

Cytogenetic analysis of meiotic cells obtained from stallion testes.

A normal course of meiosis and the associated course of spermatogenesis in males are very significant from the viewpoint of animal breeding, in particular animal reproduction. This takes on special significance when studying late-maturing animals such as horses. The aim of the study was to analyse meiotic cells, with particular consideration of synaptonemal complexes obtained from the testes of young stallions and cryptorchids, based on observations of the X-Y bivalent. The analysis was performed in successive stages of meiotic division using the FISH technique. The greatest diversity and most advanced meiotic stages were observed in the normal testis of a unilateral cryptorchid. No abnormalities were observed that could have caused cryptorchidism in the analysed horses.

FISH mapping of six genes responsible for development of the nervous and skeletal systems on donkey (Equus asinus) chromosomes.

The results obtained in the present study made it possible to place selected markers responsible for development of the nervous and skeletal systems on the physical map of the donkey genome. Fluorescence in situ hybridization (FISH) was used to localize genes such as GDF5 (15q13), FRZB (4q23.1), TWIST (1q31), PAX6 (20q25), SALL1 (24q15) and SHH (1q35) on donkey chromosomes. The identification of their localization confirmed previously proposed homologies using ZOO-FISH technique, except for FRZB and SALL1 genes. This suggests that they were affected by rearrangements that changed their localization compared to horse, and in the case of the SALL1 gene also compared to human.