Fabiana Bressan - Academia.edu (original) (raw)
Papers by Fabiana Bressan
Acta Cirurgica Brasileira, 2016
To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the ... more To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
ABSTRACT Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing ... more ABSTRACT Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing is an important tool for the improvement of gilt reproductive performance. However, there is evidence associating both flushing and EG with a disturbance in the endocrine balance that could lead to increased ovarian cysts. The aim of this study was to evaluate whether flushing or EG might affect the ovulation rate and the incidence of ovarian cysts. Seventy-one gilts were randomly distributed into 2x2 factorial design with four treatments: flushing and hormone (wFwH); no flushing and hormone (nFwH); flushing without hormone (wFnH); and neither flushing nor hormone (nFnH). Gilts were slaughtered for macroscopic and histopathological ovary examination approximately five days after artificial insemination. The characterization of these cysts was performed by optical microscopy in the following: follicular cysts (FC), luteinized cysts (LC) or cystic corpora lutea (CCL). The number of ovulations did not differ between treatments. There was no interaction between the factors in any analyzed variable. The frequency of gilts with CCL and LC was not affected by flushing and EG. No difference was found in the incidence of FC, with 12.5% and 5.88% in gilts from wFwH and nFwH treatments, respectively. There were no differences in the proportion of CCL between FC and LC (9.85 vs. 4.22 and 4.22%, respectively). In conclusion, the use of exogenous gonadotropins for second estrus synchronization in gilts, either alone or in association with dietary flushing, does not increase the incidence of ovarian cysts, nor does it decrease the ovulation rate.
Stem Cells International, 2016
Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new i... more Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine.
Genetics and Molecular Research, 2015
ABSTRACT
Pesquisa Veterinária Brasileira, 2015
Reproduction, Fertility and Development, 2015
ABSTRACT Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (... more ABSTRACT Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (NT) to oocytes and, more recently, through induction with pluripotency-related factors (iPS cells). However, the epigenetic reprogramming process that enables the derivation of both NT-derived cloned animals and iPS cells is usually incomplete, leading to unhealthy offspring and poorly reprogrammed iPS cell lines. These unfavourable outcomes result in part from abnormal genome DNA methylation that leads to aberrant gene expression patterns. For instance, differentially methylated regions (DMR) and monoalleleic expression of imprinted genes, essential for normal cellular commitment and early development, are thought to be severely disturbed by reprogramming techniques. Indeed, H19 and SNRPN, imprinted genes, were disturbed in bovine NT-derived embryos and fetuses. Herein we investigated whether the DMR and parent-of-origin expression of the imprinted genes H19 and SNRPN are also perturbed in iPS lines. To analyse the DMR methylation patterns and allelic expression of H19 and SNRPN using parental-specific polymorphisms, we derived multiple clones of bovine iPS (biPS) cells from an interspecies (Bos indicus×Bos taurus) fetal fibroblast (bFF) using transduction with a policystronic lentivirus containing mouse Oct4, Sox2 c-Myc, and Klf-4 transcription factors. The DNA methylation patterns were evaluated by bisulfite sequencing and allelic expression by designing allele-specific PCR probes. We also quantified transcript expression by RT-PCR of H19, IGF2, SNRPN, OCT4, and NANOG by normalization with 3 housekeeping genes (GAPDH, NAT1, and ACTB). The biPS lines were characterised by a high nuclear:cytoplasmic ratio, dome-shaped colonies, positive AP activity, embryoid body formation, in vitro and in vivo (teratoma) formation, and expression of pluripotency-related genes. Compared to the bFF cells, methylation analyses of H19 showed partial hypomethylation of the paternal DMR on 1 iPS cell line and partial demethylation of the CTCF-binding region in the DMR of 2 other biPS lines, indicating abnormal demethylation of 3 out of the 4 biPS lines analysed. Methylation analyses of SNRPN revealed a partial hypomethylation in the maternal DMR and partial hypermethylation of the paternal DMR in 2 iPS lines. Gene expression analyses revealed the biallelic expression of H19 and decreased global expression of both H19 and IGF2, as well as the exclusively monoallelic paternal expression and significant increase in global expression of SNRPN. Interestingly, although OCT4 was substantially overexpressed in biPS lines, we identified a hypermethylation of the CG-rich region of the OCT4 exon 1. Endogenous NANOG expression was observed in 2 biPS clones. We conclude that imprinting errors are observed in biPS clones, suggesting that these epigenetic anomalies are related to the reprogramming process and could be directly responsible for the variable phenotypes and low success rates of both cloning and iPS derivation procedures.
Reproduction, Fertility and Development, 2014
ABSTRACT Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of ... more ABSTRACT Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 10(5) cells, these cells were transduced with 50μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8ngmL(-1) polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n=10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.
Reproduction, Fertility and Development, 2015
ABSTRACT Several factors may influence transgenic animal production efficiency, and among them ge... more ABSTRACT Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP(+) cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student-Newman-Keuls test (SNK; P<0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8±2.82; AF: 10.66±0.65, CC: 3.9±1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542±497.09; AF: 9367.5±3490.9, CC: 3496±2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 - FF: 4.9±0.14 and 206.47±755; AF: 760 and 2.4±0.70±330.92; CC: 3.9±1.97, and 1418±36.06, respectively; control - FF: 0.15±0.07 and 249±6:36; AF: 0.15±0.07 588±213.54, and CC 0.05±0 214±0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.
Comparative medicine, 2011
Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neur... more Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that improve knowledge of the pathology of stroke in both human and veterinary medicine.
Reproduction, Fertility and Development, 2015
Frontiers in genetics, 2015
Animal breeders have made widespread use of assisted reproductive technologies to accelerate gene... more Animal breeders have made widespread use of assisted reproductive technologies to accelerate genetic improvement programs aimed at obtaining more, better and cheaper food products. Selection approaches have traditionally focused on Mendel's laws of inheritance using parental phenotypic characteristics and quantitative genetics approaches to choose the best parents for the next generation, regardless of their gender. However, apart from contributing DNA sequence variants, male and female gametes carry parental-specific epigenetic marks that play key roles during pre- and post-natal development and growth of the offspring. We herein review the epigenetic anomalies that are associated with artificial reproductive technologies in current use in animal breeding programs. For instance, we demonstrate that bovine embryos and fetuses derived by in vitro culture and somatic cell nuclear transfer show epigenetic anomalies in the differentially methylated regions controlling the expression...
Reproduction, Fertility and Development, 2015
Cellular reprogramming, 2012
Cell death by apoptosis is considered to be irreversible. However, reports have indicated that it... more Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 μM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of ...
Brazilian Journal of Veterinary Research and Animal Science, 2014
Acta Cirurgica Brasileira, 2016
To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the ... more To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
ABSTRACT Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing ... more ABSTRACT Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing is an important tool for the improvement of gilt reproductive performance. However, there is evidence associating both flushing and EG with a disturbance in the endocrine balance that could lead to increased ovarian cysts. The aim of this study was to evaluate whether flushing or EG might affect the ovulation rate and the incidence of ovarian cysts. Seventy-one gilts were randomly distributed into 2x2 factorial design with four treatments: flushing and hormone (wFwH); no flushing and hormone (nFwH); flushing without hormone (wFnH); and neither flushing nor hormone (nFnH). Gilts were slaughtered for macroscopic and histopathological ovary examination approximately five days after artificial insemination. The characterization of these cysts was performed by optical microscopy in the following: follicular cysts (FC), luteinized cysts (LC) or cystic corpora lutea (CCL). The number of ovulations did not differ between treatments. There was no interaction between the factors in any analyzed variable. The frequency of gilts with CCL and LC was not affected by flushing and EG. No difference was found in the incidence of FC, with 12.5% and 5.88% in gilts from wFwH and nFwH treatments, respectively. There were no differences in the proportion of CCL between FC and LC (9.85 vs. 4.22 and 4.22%, respectively). In conclusion, the use of exogenous gonadotropins for second estrus synchronization in gilts, either alone or in association with dietary flushing, does not increase the incidence of ovarian cysts, nor does it decrease the ovulation rate.
Stem Cells International, 2016
Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new i... more Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine.
Genetics and Molecular Research, 2015
ABSTRACT
Pesquisa Veterinária Brasileira, 2015
Reproduction, Fertility and Development, 2015
ABSTRACT Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (... more ABSTRACT Pluripotency reacquisition of somatic cells has been achieved through nuclear transfer (NT) to oocytes and, more recently, through induction with pluripotency-related factors (iPS cells). However, the epigenetic reprogramming process that enables the derivation of both NT-derived cloned animals and iPS cells is usually incomplete, leading to unhealthy offspring and poorly reprogrammed iPS cell lines. These unfavourable outcomes result in part from abnormal genome DNA methylation that leads to aberrant gene expression patterns. For instance, differentially methylated regions (DMR) and monoalleleic expression of imprinted genes, essential for normal cellular commitment and early development, are thought to be severely disturbed by reprogramming techniques. Indeed, H19 and SNRPN, imprinted genes, were disturbed in bovine NT-derived embryos and fetuses. Herein we investigated whether the DMR and parent-of-origin expression of the imprinted genes H19 and SNRPN are also perturbed in iPS lines. To analyse the DMR methylation patterns and allelic expression of H19 and SNRPN using parental-specific polymorphisms, we derived multiple clones of bovine iPS (biPS) cells from an interspecies (Bos indicus×Bos taurus) fetal fibroblast (bFF) using transduction with a policystronic lentivirus containing mouse Oct4, Sox2 c-Myc, and Klf-4 transcription factors. The DNA methylation patterns were evaluated by bisulfite sequencing and allelic expression by designing allele-specific PCR probes. We also quantified transcript expression by RT-PCR of H19, IGF2, SNRPN, OCT4, and NANOG by normalization with 3 housekeeping genes (GAPDH, NAT1, and ACTB). The biPS lines were characterised by a high nuclear:cytoplasmic ratio, dome-shaped colonies, positive AP activity, embryoid body formation, in vitro and in vivo (teratoma) formation, and expression of pluripotency-related genes. Compared to the bFF cells, methylation analyses of H19 showed partial hypomethylation of the paternal DMR on 1 iPS cell line and partial demethylation of the CTCF-binding region in the DMR of 2 other biPS lines, indicating abnormal demethylation of 3 out of the 4 biPS lines analysed. Methylation analyses of SNRPN revealed a partial hypomethylation in the maternal DMR and partial hypermethylation of the paternal DMR in 2 iPS lines. Gene expression analyses revealed the biallelic expression of H19 and decreased global expression of both H19 and IGF2, as well as the exclusively monoallelic paternal expression and significant increase in global expression of SNRPN. Interestingly, although OCT4 was substantially overexpressed in biPS lines, we identified a hypermethylation of the CG-rich region of the OCT4 exon 1. Endogenous NANOG expression was observed in 2 biPS clones. We conclude that imprinting errors are observed in biPS clones, suggesting that these epigenetic anomalies are related to the reprogramming process and could be directly responsible for the variable phenotypes and low success rates of both cloning and iPS derivation procedures.
Reproduction, Fertility and Development, 2014
ABSTRACT Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of ... more ABSTRACT Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 10(5) cells, these cells were transduced with 50μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8ngmL(-1) polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n=10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.
Reproduction, Fertility and Development, 2015
ABSTRACT Several factors may influence transgenic animal production efficiency, and among them ge... more ABSTRACT Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP(+) cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student-Newman-Keuls test (SNK; P<0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8±2.82; AF: 10.66±0.65, CC: 3.9±1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542±497.09; AF: 9367.5±3490.9, CC: 3496±2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 - FF: 4.9±0.14 and 206.47±755; AF: 760 and 2.4±0.70±330.92; CC: 3.9±1.97, and 1418±36.06, respectively; control - FF: 0.15±0.07 and 249±6:36; AF: 0.15±0.07 588±213.54, and CC 0.05±0 214±0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.
Comparative medicine, 2011
Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neur... more Stroke has been identified as the second leading cause of death worldwide. Stroke is a focal neurologic deficit caused by a change in cerebral circulation. The use of animal models in recent years has improved our understanding of the physiopathology of this disease. Rats and mice are the most commonly used stroke models, but the demand for larger models, such as rabbits and even nonhuman primates, is increasing so as to better understand the disease and its treatment. Although the basic mechanisms of stroke are nearly identical among mammals, we here discuss the differences between the human encephalon and various animals. In addition, we compare common surgical techniques used to induce animal models of stroke. A more complete anatomic knowledge of the cerebral vessels of various model species is needed to develop more reliable models for objective results that improve knowledge of the pathology of stroke in both human and veterinary medicine.
Reproduction, Fertility and Development, 2015
Frontiers in genetics, 2015
Animal breeders have made widespread use of assisted reproductive technologies to accelerate gene... more Animal breeders have made widespread use of assisted reproductive technologies to accelerate genetic improvement programs aimed at obtaining more, better and cheaper food products. Selection approaches have traditionally focused on Mendel's laws of inheritance using parental phenotypic characteristics and quantitative genetics approaches to choose the best parents for the next generation, regardless of their gender. However, apart from contributing DNA sequence variants, male and female gametes carry parental-specific epigenetic marks that play key roles during pre- and post-natal development and growth of the offspring. We herein review the epigenetic anomalies that are associated with artificial reproductive technologies in current use in animal breeding programs. For instance, we demonstrate that bovine embryos and fetuses derived by in vitro culture and somatic cell nuclear transfer show epigenetic anomalies in the differentially methylated regions controlling the expression...
Reproduction, Fertility and Development, 2015
Cellular reprogramming, 2012
Cell death by apoptosis is considered to be irreversible. However, reports have indicated that it... more Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 μM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of ...
Brazilian Journal of Veterinary Research and Animal Science, 2014