A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3′UTRs of candidate genes.

Establishment of embryonic stem cell (ESC) lines has been successful in mouse and human, but not in farm animals. Development of direct reprogramming technology offers an alternative approach for generation of pluripotent stem cells, applicable also in farm animals. Induced pluripotent stem cells (iPSCs) represent practically limitless, ethically acceptable, individuum-specific source of pluripotent cells that can be generated from different types of somatic cells. iPSCs can differentiate to all cell types of an organism’s body and have a tremendous potential for numerous applications in medicine, agriculture, and biotechnology. However, molecular mechanisms behind the reprogramming process remain largely unknown and hamper generation of bona fide iPSCs and their use in human clinical practice. Large animal models are essential to expand the knowledge obtained on rodents and facilitate development and validation of transplantation therapies in preclinical studies. Additionally, transgenic animals with special traits could be generated from genetically modified pluripotent cells, using advanced reproduction techniques. Despite their applicative potential, it seems that iPSCs in farm animals haven’t received the deserved attention. The aim of this review was to provide a systematic overview on iPSC generation in the most important mammalian farm animal species (cattle, pig, horse, sheep, goat, and rabbit), compare protein sequence similarity of pluripotency-related transcription factors in different species, and discuss potential uses of farm animal iPSCs. Literature mining revealed 32 studies, describing iPSC generation in pig (13 studies), cattle (5), horse (5), sheep (4), goat (3), and rabbit (2) that are summarized in a concise, tabular format.

BackgroundCryptorchidism is the most frequent congenital disorder in male children; however the genetic causes of cryptorchidism remain poorly investigated. Comparative integratomics combined with systems biology approach was employed to elucidate genetic factors and molecular pathways underlying testis descent.MethodsLiterature mining was performed to collect genomic loci associated with cryptorchidism in seven mammalian species. Information regarding the collected candidate genes was stored in MySQL relational database. Genomic view of the loci was presented using Flash GViewer web tool (http://gmod.org/wiki/Flashgviewer/). DAVID Bioinformatics Resources 6.7 was used for pathway enrichment analysis. Cytoscape plug-in PiNGO 1.11 was employed for protein-network-based prediction of novel candidate genes. Relevant protein-protein interactions were confirmed and visualized using the STRING database (version 9.0).ResultsThe developed cryptorchidism gene atlas includes 217 candidate loci (genes, regions involved in chromosomal mutations, and copy number variations) identified at the genomic, transcriptomic, and proteomic level. Human orthologs of the collected candidate loci were presented using a genomic map viewer. The cryptorchidism gene atlas is freely available online: http://www.integratomics-time.com/cryptorchidism/. Pathway analysis suggested the presence of twelve enriched pathways associated with the list of 179 literature-derived candidate genes. Additionally, a list of 43 network-predicted novel candidate genes was significantly associated with four enriched pathways. Joint pathway analysis of the collected and predicted candidate genes revealed the pivotal importance of the muscle-contraction pathway in cryptorchidism and evidence for genomic associations with cardiomyopathy pathways in RASopathies.ConclusionsThe developed gene atlas represents an important resource for the scientific community researching genetics of cryptorchidism. The collected data will further facilitate development of novel genetic markers and could be of interest for functional studies in animals and human. The proposed network-based systems biology approach elucidates molecular mechanisms underlying co-presence of cryptorchidism and cardiomyopathy in RASopathies. Such approach could also aid in molecular explanation of co-presence of diverse and apparently unrelated clinical manifestations in other syndromes.

The article presents multi-species, genome-wide, comparative approach to review male fertility-associated loci to contribute to the development of new genetic markers that could be of interest for functional studies and have the potential to be implemented in farm animal breeding programmes. We reviewed 835 male fertility-associated candidate loci from seven species and presented them as bovine orthologues where possible. The candidate loci were identified exploiting seven different research approaches: (i) data from animal models: mouse transgenics and knock-outs (569 genes) and random chemical mutagenesis of mouse genome (31); (ii) animal QTL (69); (iii) genes differentially expressed between fertile and subfertile phenotype in humans and mouse (95); (iv) DNA sequence variations that show specific allele-phenotype interactions (43 in human and 13 in farm animals); (v) germ line-specific small non-coding RNAs (47); (vi) testes expressed genes controlling complex differentiation process of mammalian spermatogenesis (6); and (vii) epigenetically regulated genes (4). According to the number of different research approaches reporting effects of individual genes, we selected 33 most promising candidate genes, which were further in silico analysed for expression levels in testes, genetic variability and top biological functions in functional networks. The aim of this study was to review systematically male fertility-associated candidate loci using integrated information from different study approaches and species, which will further facilitate development of novel genetic markers for selection towards improved fertility in domestic animals.

ABSTRACT. Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormonesupplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other J. Ogorevc and P. Dovč 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

BackgroundForkhead box P3+ (FOXP3+) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1–3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2).ResultsUsing an activator-domain fusion based dCas9 transcription activator, robust upregulation of FOXP3 was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on FOXP3 gene upregulation. Simultaneous targeting of FOXP3 and EOS, a transcription factor known to act in concert with FOXP3 in initiating a Treg phenotype, resulted in upregulation of FOXP3 downstream genes CD25 and TNFR2. When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells.ConclusionsTransfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR–dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.

The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR) was quantified in mammary tissue-derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up-regulation of ESR1 (1.36- to 12.35-fold) and in down-regulation of PGR (-2.53- to -3.62-fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER-α) positive cells do not exclusively belong to the luminal lineage and that ER-α and PGR can be expressed individually or co-expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone-responsive and represent a useful surrogate for mammary tissue in research experiments.

The objective of this study was morphological and functional characterization of cells from the primary cell culture developed from lactating goat mammary gland, focusing on distribution of lineage-specific markers. Primary cells were grown on a thin layer of basement membrane matrix, a growth surface that resembles in vivo conditions. The cells in adherent conditions rapidly proliferated and showed cobblestone morphology, typical for epithelial cells. Under non-adherent conditions, goat mammary cells formed spherical, acini-like structures that resembled alveoli of lactating mammary gland. Immunofluorescence and RNA sequencing were employed to determine expression of lineage-specific markers. Presence of markers cytokeratin 14 and 18, integrin alpha 6, vimentin, estrogen receptor, smooth muscle actin, and cytokeratin 5 was detected using immunofluorescence. The greatest expression was observed for markers typical for myoepithelial cells, luminal cells, and mesenchymal cells. Based on our characterization, we can conclude that established primary culture was composed of mainly epithelial and stromal cells. These findings demonstrate that primary mammary cells express some of the most important functional and biochemical markers needed for their characterization. First, they grow in the characteristic cobblestone morphology of epithelial cells. Second, they express classical cytoplasmic network of cytokeratin fibers. Third, they express markers typical of mammary parenchyma and stroma. The established cell culture represents a good in vitro model for studies of mammary gland development, differentiation, and lactation. We suggest that herein revealed lineage markers are suitable for characterization of mammary cells of goat and possibly other mammalian species.