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Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.

Objective Complete mtDNA D-loop sequences of four Thai indigenous chicken varieties, including Pra-dhu-hang-dam (PD), Leung-hang-khao (LK), Chee (CH), and Dang (DA) were explored for genetic diversity and relationships with their potential ancestor and possible associates to address chicken domestication in Thailand. Methods A total of 220 complete mtDNA D-loop sequences of the four Thai indigenous chicken varieties were obtained by Sanger direct sequencing of polymerase chain reaction amplicons of 1,231 to 1,232 base pair in size. A neighbor-joining dendrogram was constructed with reference complete mtDNA D-loop sequences of Red Junglefowl (RJF) and those different chicken breeds available on National Center for Biotechnology Information database. Genetic diversity indices and neutrality test by Tajima's D test were performed. Genetic differences both within and among populations were estimated using analysis of molecular variance (AMOVA). Pairwise fixation index (FST) was conducted to evaluated genetic relationships between these varieties. Results Twenty-three identified haplotypes were classified in six haplogroups (A-E and H) with the majority clustered in haplogroup A and B. Each variety was in multiple haplogroups with haplogroups A, B, D, and E being shared by all studied varieties. The averaged haplotype and nucleotide diversities were, respectively 0.8607 and 0.00579 with non-significant Tajima's D values being observed in all populations. Haplogroup distribution was closely related to that of RJF particularly Gallus gallus gallus (G. g. gallus) and G. g. spadiceus. As denoted by AMOVA, the mean diversity was mostly due to within-population variation (90.53%) while between-population variation (9.47%) accounted for much less. By pairwise FST, LK was most closely related to DA (FST = 0.00879) while DA was farthest from CH (FST = 0.24882). Conclusion All 4 Thai indigenous chickens are in close relationship with their potential ancestor, the RJF. A contribution of shared, multiple maternal lineages was in the nature of these varieties, which have been domesticated under neutral selection.

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P

Zebrafish (Danio rerio) is an established animal model to study developmental biology as well as a wide array of human diseases. Here we describe a protocol for somatic cell nuclear transfer (SCNT). This protocol can be used to introduce genetic modifications in zebrafish and for the study of cell plasticity.

Zebrafish (Danio rerio) is an established animal model to study developmental biology as well as a wide array of human diseases. Here we describe a protocol for somatic cell nuclear transfer (SCNT). This protocol can be used to introduce genetic modifications in zebrafish and for the study of cell plasticity.

This report characterized seven cases of canine retrobulbar lymphoma that have been diagnosed during 2008 to 2014 by immunophenotyping of CD3 and Pax5. Classification of lymphoma were performed according to the revised WHO guidelines. Four retrobulbar lymphomas were of T-cell origin, while the others were of B-cell. Out of 7 cases, four subtypes were diagnosed in this study; T-cell-rich large B-cell lymphoma (3/7), T-cell lymphoblastic lymphoma (2/7), peripheral T-cell lymphoma (1/7), and cutaneous nonepitheliotropic lymphoma (1/7). T-cell-rich large B-cell lymphoma was found to be the most frequent subtype found.Keywords: Dog, Immunohistochemistry, Lymphoma, Retrobulbar

Avian primordial germ cells (PGCs) are destined to migrate a long distance from their extra embryonic region via the vascular system to the gonadal ridges where they form the germ cells. Although PGC migration is crucial for a genetic continuation to the next generation, the factors and mechanisms that control their migration remain largely unknown. In the present study the chemotactic effect of stem cell factor (SCF) was examined on chicken blood circulating PGCs (cPGC), employing 3D chemotaxis slides and time-lapsed imaging analyses as an in vitro study model. Upon in vitro exposure to an SCF gradient, 77.1% (54 out of 70) of cPGCs showed a clear response, of which 48.1% (26 out of 54) polarized with the consecutive formation of a persistent membrane protrusion and significant directional migration towards the gradient and the others showed transient membrane protrusions. In contrast, the controls and apparently SCF unresponsive cPGCs and c-kit-negative red blood cells (RBCs) showed only cytoplasmic cycling with random formations of membrane blebbing and no directional migration. Significant (p < 0.05) differences between the SCF-treated and control cPGCs and RBCs were found in the migration parameters of eccentricity, accumulated and Euclidean distances, and migration velocity. The SCF-treated PGCs also revealed a chemotactic response, as judged by their significant displacement of center of mass and Rayleigh test. Complete inhibition of all the SCF-induced responses in PGCs was found following pretreatment of the cPGCs with 10 µM of the c-kit inhibitor, STI57l, prior to SCF exposure. In addition, cPGCs were found to be positive for c-kit expression using a polyclonal goat anti-mouse c-kit primary antibody, suggesting that the cPGCs were capable of SCF sensing and the potential involvement of SCF/c-kit in the chemotactic migration. Therefore, SCF is suggested to function as a chemoattractant in the migration of chicken cPGC.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

The success of nuclear reprogramming following somatic cell nuclear transfer (SCNT) is thought to depend on factors present in the egg. Little is known about the role - if any - played by the somatic cell type on the outcome of the procedure. We tested whether cells of different lineages might have different capacities for reprogramming following SCNT, comparing cells isolated from five different tissues of transgenic zebrafish for their developmental potential when used as SCNT donor cells. We used transgenic zebrafish lines expressing green fluorescence protein under an endogenous tissue-specific promoter: HGn62A-skin, HGn28A-skin, HGn8E-heart, HG21C-fin and notochord and HGn30A-hatch gland. We analyzed the efficiency of cloning, as measured by reconstructed embryos that developed up to the hatched-fry stage. Specifically, donor cells of fin and notochord origin yielded the best rate of cloned fish production. All of the other cell types used were capable of producing cloned fish, albeit with significantly lower efficiency. These results indicate that the type of zebrafish cells used for SCNT can influence the outcome of the procedure. Future epigenetic analysis of these cells will help determine specific chromatin profiles in somatic cells that have an impact on nuclear reprogramming procedures.

We describe the characterization of maturation-promoting factor (MPF) in zebrafish eggs and used different defined conditions to maintain its activity in vitro. MPF activity levels are high in freshly ovulated mature eggs and decline rapidly within 5 min after either fertilization or parthenogenetic activation. The MPF activity of eggs matured in vitro declines faster when the eggs are incubated in Hank's culture medium supplemented with 0.5% BSA (H-BSA) than when incubated in Chinook salmon ovarian fluid (CSOF). MPF activity in nonactivated, aged eggs remains high in H-BSA supplemented with 75 microM MG132 or 10 mM caffeine, but neither MG132 nor caffeine can sustain high MPF activity in activated eggs. MG132-treated eggs showed delayed completion of metaphase and extrusion of the second polar body. Nuclear staining of the activated eggs confirmed the correlation between their cell cycle stage and MPF activity at each time point. An embryotoxic effect was found when matured eggs were held in 100 microM of MG132 or 20 mM caffeine for 1 h. Calcium-depleted medium and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid also showed detrimental effects on the embryos. Conversely, nonactivated, aged matured eggs maintained high MPF activity and developmental potential when CSOF was used as a holding medium.

Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures.

Technical modifications of existing methods lead to a somatic cell nuclear transfer method in the zebrafish, which yields adult cloned fish with healthy offspring that carry donor traits. We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.

The use of Zebrafish as a model-organism is growing exponentially. Somatic Cell Nuclear Transfer could potentially enhance the way gene modifications are done and facilitate the selection and preservation of specific strains and mutant lines. However, since first reported in 2002, SCNT failed to be implemented widely. The reason for the lack of widespread use is likely the difficulty to perform the protocol as was described. This chapter addresses all the critical parameters in the procedure including handling of the recipient eggs, donor cells, and care of the reconstructed embryos. We describe a novel protocol that overcomes common hindrances and incorporates a new approach for enucleation and cell transfer, parameters that can greatly influence SCNT success. We predict that the simplicity of this protocol will entice other laboratories to use it.

Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures.

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (p.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

Trimethylation of histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes andin vitro-produced embryos were collected at different stages of development. Polycomb gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts forEZH2,EED, andSUZ12were detected at all stages analyzed, withEZH2levels being the highest of the three at early stages of development. By the time the embryo reached the blastocyst stage, the level ofPcGgene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression ofEZH2at all stages while nuclear localizedEEDandSUZ12were only evident at the morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was the highest in immature oocytes, which steadily decreased after fertilization to reach a nadir at the eight-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development.