Search

As nucleic acid (NA) technologies continue to revolutionize medicine, new delivery vehicles are needed to effectively transport NA cargoes into cells. Uniform and length-tunable nanofiber micelleplexes have recently shown promise as versatile polymeric delivery vehicles for plasmid DNA, however the effects of several key parameters on micelleplex transfection and stability remain unknown. In this work, we compare poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC-b-PDMAEMA) nanofiber micelleplexes to nanosphere micelleplexes and PDMAEMA polyplexes, examining the effects of complexation buffer, the temporal and serum stability of nanofiber micelleplexes, as well as the effects of cell density, cell type, and polymer DPnupon transfection efficiency and cell viability. These studies are vital for understanding the formation and biological activity of micelleplexes in more detail and should inform the future design of more advanced polymeric NA delivery systems.

Micelleplexes show great promise as effective polymeric delivery systems for nucleic acids. Although studies have shown that spherical micelleplexes can exhibit superior cellular transfection to polyplexes, to date there has been no report on the effects of micelleplex morphology on cellular transfection. In this work, we prepared precision, length-tunable poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC16-b-PDMAEMA131) nanofiber micelleplexes and compared their properties and transfection activity to those of the equivalent nanosphere micelleplexes and polyplexes. We studied the DNA complexation process in detail via a range of techniques including cryo-transmission electron microscopy, atomic force microscopy, dynamic light scattering, and ζ-potential measurements, thereby examining how nanofiber micelleplexes form, as well the key differences that exist compared to nanosphere micelleplexes and polyplexes in terms of DNA loading and colloidal stability. The effects of particle morphology and nanofiber length on the transfection and cell viability of U-87 MG glioblastoma cells with a luciferase plasmid were explored, revealing that short nanofiber micelleplexes (length < ca. 100 nm) were the most effective delivery vehicle examined, outperforming nanosphere micelleplexes, polyplexes, and longer nanofiber micelleplexes as well as the Lipofectamine 2000 control. This study highlights the potential importance of 1D micelleplex morphologies for achieving optimal transfection activity and provides a fundamental platform for the future development of more effective polymeric nucleic acid delivery vehicles.

Mesenchymal stem cells (MSCs) are multilineage cells able to differentiate into other cell types. MSCs derived from bone marrow or compact bones are the most accessible stem cells used in tissue engineering. Therefore, the aim of this study was to isolate, characterize and cryopreserve MSCs of endangered Oravka chicken breed. MSCs were obtained from compact bones of the femur and tibiotarsus. MSCs were spindle-shaped and were able to differentiate into osteo-, adipo-, and chondrocytes under the specific differentiation conditions. Furthermore, MSCs were positive for surface markers such as CD29, CD44, CD73, CD90, CD105, CD146 and negative for CD34CD45 by flow cytometry. Moreover, MSCs demonstrated high positivity of “stemness” markers aldehyde dehydrogenase, alkaline phosphatase as well as for intracellular markers vimentin, desmin, α-SMA. Subsequently, MSCs were cryopreserved using 10% dimethyl sulfoxide in liquid nitrogen. Based on the results from the viability, phenotype, and ultrastructure assessment we can concluded that the MSCs were not negatively affected by the cryopreservation. Finally, MSCs of endangered Oravka chicken breed were successfully stored in animal gene bank, thus making them a valuable genetic resource.

Ophthalmic researchers and clinicians arguably have led the way for safe, effective gene therapy, most notably with adeno-associated viral gene supplementation in the treatment for patients with Leber congenital amaurosis type 2 with mutations in the RPE65gene. These successes notwithstanding, most other genetic retinal disease will be refractory to supplementation. The ideal gene therapy approach would correct gene mutations to restore normal function in the affected cells. Gene editing in which a mutant allele is inactivated or converted to sequence that restores normal function is hypothetically one such approach. Such editing involves site-specific digestion of mutant genomic DNA followed by repair. Previous experimental approaches were hampered by inaccurate and high rates of off-site lesioning and by overall low digestion rates. A new tool, clustered regularly interspaced short palindromic repeats coupled with the nuclease Cas9, may address both shortcomings. Some of the many challenges that must be addressed in moving clustered regularly interspaced short palindromic repeats coupled with the nuclease Cas9 therapies to the ophthalmic clinic are discussed here.

The aim of the study was to define ultrastructural changes in the ovarian cyclic corpus luteum (CL) of dairy cows with different body condition score (BCS, a 5-point scale). The ovaries were recovered from slaughtered Holstein cows of BCS1 (emaciation; n= 4), BCS2 (tendency to emaciation; n=4), BCS3 (average body condition; n= 4), BCS4 (tendency to fattening; n=4) and BCS5 (fattening; n=4). Average ages of cows were: 6.2, 4.1, 5.7, 5.5 and 6.36 years for BCS1, BCS2, BCS3, BCS4 and BCS5, respectively. For electron microscopy, functioning CL at the mid-luteal phase were fixed in the aldehyde solution and post-fixed in 1% OsO4 solution. For lipid visualization the samples were soaked in 1% p-phenylenediamine solution in acetone. Durcupan ultrathin sections (90 nm) were contrasted with uranyl acetate and lead citrate and viewed under Jeol transmission electron microscope. Occurrence of cell organelles was determined by counting the relative volume of individual cellular components using the modified universal test network. Luteal regression of cyclic CL was characterized by atrophy of lutein cells, condensation of organelles and cytoplasmic inclusions, increase in relative volumes of vacuoles, lysosomes, peroxisomes, autophagosomes and lipids. There were significant differences (p<0.05) among BCS1, BCS2, BCS4 and BCS5 cows in the occurrence of lipids, vacuoles, lysosomes and autophagosomes in the lutein cells compared to BCS3 cows. Cows with BCS2 or BCS1 had higher relative volume of lipid droplets, whilst the BCS4 or BCS5 cows had increased occurrence (relative volume) of vacuoles, lysosomes, peroxisomes and autolysosomes. These results show that luteolysis of cyclic CL of cows has different patterns in each BCS group; in the emaciated and fattened cows luteolysis starts earlier than in cows with average body condition.

The objective of this study was to assess the effect of different diluent type and storage time on the quality of rooster insemination doses in terms of the spermatozoa motility and apoptosis (in vitro) and spermatozoa fertility potential (in vivo). The motility parameters were significantly (P< 0.001) higher in the spermatozoa diluted in saline solution than those diluted in avian diluent at each storage time (0.5, 1 and 2 hours). The proportion of apoptotic spermatozoa was significantly (P< 0.05) higher in the semen samples diluted in the avian diluent in comparison to the semen samples diluted in the saline solution after 1 hour of storage. The proportion of dead spermatozoa was significantly (P< 0.05) higher in avian diluent in comparison to the semen samples diluted in the saline solution after 2 hours of storage. The fertility of spermatozoa diluted in the avian diluent significantly (P< 0.01) decreased 1 hour after the ejaculate collection. In conclusion, saline solution maintained better quality of rooster spermatozoa in terms of their motility values and the proportion of apoptotic and dead cells (in vitro) than the commercial avian diluent when stored at 4 °C for 2 hours. According to in vivoresults, the saline solution seems to be suitable for semen dilution at low temperature, since similar fertility results were obtained over the whole storage time. Thus the commercial avian diluent could be replaced by the saline solution under certain conditions. Furthermore, the saline solution is cheaper and more accessible for practical use than the commercial avian diluent.

3D bioprinting—a type of additive manufacturing—can create 3D tissue constructs resembling in vivotissues. Here, we present a protocol for 3D printing neural tissues using Axolotl Biosciences’ fibrin-based bioink and the CELLINK BIO X bioprinter with a pneumatic printhead. This workflow can be applied to printing 3D tissue models using a variety of cell lines and any chemically crosslinked bioink. These 3D-printed tissue models can be used for applications such as drug screening and disease modeling in vitro.

It was previously documented that cyclic AMP (cAMP)-dependent intracellular mechanisms can be involved in control of reproductive processes, and pharmacological regulators of these mechanisms could be practically used to improve rabbit fertility (SIROTKIN et al. 2008; SIROTKIN et al. 2010a; CHRENEK et al. 2012). Changes in fertility could be due to changes in oviductal functions. The aim of our study was to examine the involvement of cAMP-dependent intracellular mechanisms in control of oviductal cell functions, in particular the influence of dbcAMP, a cAMP agonistic analogue, on proliferation and apoptosis of cultured oviductal cells. For this purpose, we compared the expression of markers of proliferation (PCNA, cyclin B1) and apoptosis (bax and bcl-2) in the oviduct epithelial cells isolated from rabbits, whose ovarian and oviductal cycle was induced by gonadotropins alone or in combination with dbcAMP (50 μg/animal) by using immunocytochemistry. It was observed that dbcAMP administration caused an increase in the proportion of cells containing PCNA, but not cyclin B1, bax or bcl-2. Higher expression of PCNA, but not cyclin B1, in the dbcAMP-treated group suggests that the dbcAMP administration can stimulate oviductal cell proliferation, probably promoting transition of cells from G0 to G1 and S-phase of the cell cycle. No influence of dbcAMP administration on regulators and markers of apoptosis (pro-apoptotic – bax and anti-apoptotic – bcl-2) suggests that dbcAMP is probably not involved in the control of apoptosis in rabbit oviduct epithelial cells. The involvement of cAMP-dependent intracellular mechanisms in control of oviduct functions is assumed in this study. This is the first demonstration that dbcAMP can stimulate proliferation of the oviduct epithelial cells without influencing their apoptosis.

The aim of our study is to examine the influence of administration of 3-isobutyl-l-methyl-xanthine (IBMX), inhibitor of cAMP and cGMP phosphodiesterases on ovarian functions (folliculogenesis, atresia and luteogenesis), as well as on some reproductive parameters in rabbits whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by PMSG followed by hCG administration. Experimental animals received 20IU/kg PMSG and 35IU/kg hCG together with IBMX (at 5, 25 or 50 µg/animal). After ovulation and mating, the animals were sacrificed. Histological slides of ovaries were prepared, and the presence of follicles and different stages of luteinisation and atresia were evaluated by light microscopy. The pronuclear stage eggs were flushed out from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived embryos reaching blastocyst stage were determined. Administration of IBMX was able to increase the proportion of luteinised follicles. Furthermore, IBMX treatment promoted occurrence of atresia in the remaining follicles after the gonadotropin treatment. Finally, IBMX increased the number of Corpora lutea, number of harvested zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that IBMX can enhance the stimulatory effect of gonadotropins on the rabbit ovarian follicle luteinisation, atresia, ovulation, zygote and embryo yield and development. Furthermore, they confirm the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of IBMX in improvement of farm animal reproduction and fertility.

During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin+ lymph sacs and cardinal veins, but not in podoplanin−/− embryos. Thus, podoplanin−/− mice develop a “nonseparation” phenotype, characterized by a blood-filled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin-blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p<0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not siginificantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.

Abstract Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19–20 h after hCG injection. About 5 μg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3′ flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 μg/mL rhFVIII in milk, with an activity of 0.594 lU/mL. The DM founder female produced 118 μg/mL rhFVIII, with activity values of 18 lU/mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19–20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94–96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p<0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi-and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p<0.001) and lower total cell number (p<0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Stromal–epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization. Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease. Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment. Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance.

Nickel (Ni) and zinc (Zn) are trace elements present at low concentrations in agroecosystems. Nickel, however, may have toxic effects on living organisms and is often considered as a contaminant. This study reports the effect of peroral administrated Ni or a combination of Ni and Zn on femoral bone structure in rabbits.