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Simoni, Michael K., Sydnie Swanson, Monica Mainigi, and Kellie Jurado. "22732 Impact of Type-I Interferon Manipulation During Embryo Implantation and Placentation." Journal of Clinical and Translational Science 5, s1 (March 2021): 94–95. http://dx.doi.org/10.1017/cts.2021.644.
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ABSTRACT IMPACT: This research will promote understanding the role of the Type-I Interferon signaling pathway during embryo implantation, potentially leading to a new diagnostic or treatment target in early pregnancy failure. OBJECTIVES/GOALS: Studies suggest interferon signaling regulation is tightly balanced between physiologic and pathophysiologic growth in early pregnancy. We propose to determine the impact of interferon-mediated inflammation on embryo implantation and early pregnancy failure in normal conditions and chronic inflammatory diseases in a novel mixed-mouse model. METHODS/STUDY POPULATION: To probe the role of type-I interferons (IFNs) in implantation, we will utilize a mouse model and non-surgically transfer both Ifnar1-/- and Ifnar1-/+ embryos into an immune-competent pseudopregnant wild-type female recipient. This will allow analysis of a litter with distinct genotypes within the same, immune-competent, uterine environment. Type-I IFN stimulation will be systemically induced with Poly-(I:C) at various time points around implantation. A similar approach will be used in mouse models of chronic inflammatory disease states associated with early pregnancy loss (e.g. systemic lupous erythematous). With this model, we will be able to control for deficiencies in maternal immune response to specifically determine the embryonic response to inflammation during implantation and development. RESULTS/ANTICIPATED RESULTS: We anticipate the Ifnar1-/+ embryos - those able to respond to Type-I IFN - and their surrounding implantation sites will exhibit more maternal-fetal barrier dysfunction in the form of impaired trophoblast fusion, improper formation of the microvascular architecture, and increased permeability of the maternal-fetal barrier, compared to embryos unable to respond to IFN. We will also conduct similar analyses in mouse models of chronic inflammatory diseases. We hypothesize these mice to have baseline endometrial inflammation that stimulated the IFN-pathway in IFN-capable embryos, producing breakdown of the maternal-fetal barrier. In these mice, we predict Ifnar1-/- embryos will show improved molecular outcomes when compared to Ifnar1-/+ embryos, and thus improved associated pregnancy outcomes. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work can insight into the immunological mechanisms that govern embryo implantation and early placentation. This could provide more pointed means for management and intervention of early pregnancy failure and/or disease states that are commonly associated with poor reproductive outcomes.
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Kumar, Akash, Kate Im, Milena Banjevic, Pauline C. Ng, Tate Tunstall, Geronimo Garcia, Luisa Galhardo, et al. "Whole-genome risk prediction of common diseases in human preimplantation embryos." Nature Medicine 28, no. 3 (March 2022): 513–16. http://dx.doi.org/10.1038/s41591-022-01735-0.
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AbstractPreimplantation genetic testing (PGT) of in-vitro-fertilized embryos has been proposed as a method to reduce transmission of common disease; however, more comprehensive embryo genetic assessment, combining the effects of common variants and rare variants, remains unavailable. Here, we used a combination of molecular and statistical techniques to reliably infer inherited genome sequence in 110 embryos and model susceptibility across 12 common conditions. We observed a genotype accuracy of 99.0–99.4% at sites relevant to polygenic risk scoring in cases from day-5 embryo biopsies and 97.2–99.1% in cases from day-3 embryo biopsies. Combining rare variants with polygenic risk score (PRS) magnifies predicted differences across sibling embryos. For example, in a couple with a pathogenic BRCA1 variant, we predicted a 15-fold difference in odds ratio (OR) across siblings when combining versus a 4.5-fold or 3-fold difference with BRCA1 or PRS alone. Our findings may inform the discussion of utility and implementation of genome-based PGT in clinical practice.
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Feuer, Sky K., Xiaowei Liu, Annemarie Donjacour, Wingka Lin, Rhodel K. Simbulan, Gnanaratnam Giritharan, Luisa Delle Piane, et al. "Use of a Mouse In Vitro Fertilization Model to Understand the Developmental Origins of Health and Disease Hypothesis." Endocrinology 155, no. 5 (May 1, 2014): 1956–69. http://dx.doi.org/10.1210/en.2013-2081.
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The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.
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Rosner, Margit, Manuel Reithofer, Dieter Fink, and Markus Hengstschläger. "Human Embryo Models and Drug Discovery." International Journal of Molecular Sciences 22, no. 2 (January 11, 2021): 637. http://dx.doi.org/10.3390/ijms22020637.
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For obvious reasons, such as, e.g., ethical concerns or sample accessibility, model systems are of highest importance to study the underlying molecular mechanisms of human maladies with the aim to develop innovative and effective therapeutic strategies. Since many years, animal models and highly proliferative transformed cell lines are successfully used for disease modelling, drug discovery, target validation, and preclinical testing. Still, species-specific differences regarding genetics and physiology and the limited suitability of immortalized cell lines to draw conclusions on normal human cells or specific cell types, are undeniable shortcomings. The progress in human pluripotent stem cell research now allows the growth of a virtually limitless supply of normal and DNA-edited human cells, which can be differentiated into various specific cell types. However, cells in the human body never fulfill their functions in mono-lineage isolation and diseases always develop in complex multicellular ecosystems. The recent advances in stem cell-based 3D organoid technologies allow a more accurate in vitro recapitulation of human pathologies. Embryoids are a specific type of such multicellular structures that do not only mimic a single organ or tissue, but the entire human conceptus or at least relevant components of it. Here we briefly describe the currently existing in vitro human embryo models and discuss their putative future relevance for disease modelling and drug discovery.
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Rosner, Margit, Manuel Reithofer, Dieter Fink, and Markus Hengstschläger. "Human Embryo Models and Drug Discovery." International Journal of Molecular Sciences 22, no. 2 (January 11, 2021): 637. http://dx.doi.org/10.3390/ijms22020637.
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For obvious reasons, such as, e.g., ethical concerns or sample accessibility, model systems are of highest importance to study the underlying molecular mechanisms of human maladies with the aim to develop innovative and effective therapeutic strategies. Since many years, animal models and highly proliferative transformed cell lines are successfully used for disease modelling, drug discovery, target validation, and preclinical testing. Still, species-specific differences regarding genetics and physiology and the limited suitability of immortalized cell lines to draw conclusions on normal human cells or specific cell types, are undeniable shortcomings. The progress in human pluripotent stem cell research now allows the growth of a virtually limitless supply of normal and DNA-edited human cells, which can be differentiated into various specific cell types. However, cells in the human body never fulfill their functions in mono-lineage isolation and diseases always develop in complex multicellular ecosystems. The recent advances in stem cell-based 3D organoid technologies allow a more accurate in vitro recapitulation of human pathologies. Embryoids are a specific type of such multicellular structures that do not only mimic a single organ or tissue, but the entire human conceptus or at least relevant components of it. Here we briefly describe the currently existing in vitro human embryo models and discuss their putative future relevance for disease modelling and drug discovery.
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Bowley, George, Timothy JA Chico, Jovana Serbanovic-Canic, and Paul C. Evans. "Quantifying endothelial cell proliferation in the zebrafish embryo." F1000Research 10 (October 11, 2021): 1032. http://dx.doi.org/10.12688/f1000research.73130.1.
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Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.
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Swanson, W. F., H. L. Bateman, J. Newsom, V. A. Conforti, J. R. Herrick, C. A. Lambo, M. E. Haskins, et al. "55 PROPAGATION OF MULTIPLE CAT HEREDITARY DISEASE MODELS FOLLOWING ASSISTED REPRODUCTION WITH FROZEN SEMEN AND EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 139. http://dx.doi.org/10.1071/rdv24n1ab55.
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Domestic cats are invaluable research models for the study of hereditary diseases that affect both cats and humans. By necessity, most cat models are maintained as living populations; however, semen and embryo cryopreservation could provide a cost-effective alternative for model conservation if viable offspring can be readily produced with assisted reproductive techniques such as IVF, embryo transfer (ET) and AI. In our earlier research, semen and IVF-derived embryos representing 24 cat models at 7 veterinary/medical schools were frozen for liquid nitrogen storage. Our objectives in this study were to (1) assess the application of assisted reproduction using frozen semen or embryos for producing pregnancies and viable kittens in several cat models and (2) provide our university collaborators with rederived model offspring. Five cat models (i.e. spinal muscular atrophy, SMA; porphyria, POR; Chediak-Higashi syndrome, CHS; progressive retinal atrophy, PRA; and hypertrophic cardiomyopathy, HCM) were selected for propagation based on investigator research needs. For 3 models (SMA, POR and CHS), semen from affected males, frozen as pellets in Test Egg Yolk medium (with 4% glycerol), was thawed and used to inseminate (5 × 105 motile sperm mL–1) oocytes from eCG/hCG-treated queens. Resulting embryos (2- to 8-cell stage) were transferred laparoscopically (3–5 embryos/recipient) into the oviducts of anestrual queens synchronized with eCG/pLH. For PRA, IVF embryos from affected cats were frozen in 1.5 M ethylene glycol and later thawed for transfer into eCG/pLH-synchronized queens and for HCM, frozen semen from a carrier male was used for laparoscopic oviducal insemination (1 × 106 to 4 × 106 motile sperm/AI) of eCG/pLH-treated females. Ultrasonography was conducted approximately 21 days post-ET or -AI for pregnancy diagnosis. Following IVF with frozen-thawed semen, embryos were produced in all 3 models but overall fertilization success was low (21%, 34/164). All (5/5) recipients became pregnant after ET, giving birth to a total of 11 offspring (6 viable, 5 stillborn). Following frozen ET (PRA), most (3/5) recipients became pregnant with 6 kittens carried to term (3 viable, 3 stillborn), whereas with frozen semen AI (HCM), most (4/7) females conceived with the subsequent birth of 22 kittens (all viable). All surviving offspring (n = 25) for the 5 disease models were distributed to collaborating veterinary schools to reestablish breeding colonies or for ongoing studies. These results indicate that assisted reproduction using frozen semen or embryos may be applied effectively with specific cat models to propagate desired lineages or reestablish research colonies, although some other models have proven more difficult to reproduce. These findings validate our contention that cryopreservation and assisted reproduction may be used to manage and conserve these irreplaceable cat disease models. Funded by NIH grants RR015338, RR02512, HL093603 and HD39888.
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Tobia, Chiara, Daniela Coltrini, Roberto Ronca, Alessandra Loda, Jessica Guerra, Elisa Scalvini, Francesco Semeraro, and Sara Rezzola. "An Orthotopic Model of Uveal Melanoma in Zebrafish Embryo: A Novel Platform for Drug Evaluation." Biomedicines 9, no. 12 (December 10, 2021): 1873. http://dx.doi.org/10.3390/biomedicines9121873.
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Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy.
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Gregg, K., S. Chen, S. Sadeghieh, T. Guerra, T. Xiang, J. Meredith, and I. Polejaeva. "149 RISK ASSESSMENT OF INFECTIOUS DISEASE TRANSMISSION VIA IN VITRO EMBRYO PRODUCTION USING SOMATIC CELL NUCLEAR TRANSFER." Reproduction, Fertility and Development 21, no. 1 (2009): 174. http://dx.doi.org/10.1071/rdv21n1ab149.
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Somatic cell nuclear transfer (SCNT) technology is a powerful tool for preservation and propagation of superior genetics of livestock animals. Bovine oocytes derived from abattoirs are usually used in SCNT embryo production. The puncture of the zona pellucida during the nuclear transfer process has raised additional concerns about the risk of disease transmission through application of this new technology. The objective of this study was to use bovine viral diarrhea virus (BVDV) as a model to perform a comprehensive risk assessment on infectious disease transmission in the SCNT system. Thirteen batches of cumulus–oocyte complexes (COC; n = 550) were collected from several abattoirs over 6 months. Two hundred were tested for BVDV before cumulus cell removal. Cumulus cells were removed from the other 350 COC by gentle vortexing in 0.1% hyaluronidase in HEPES-M199 with Hanks’ salts. The cumulus-cell-free oocytes (CFO) were then washed 3 times with FBS-free HEPES-M199 with Hanks’ salts containing penicillin-streptomycin solution (5 μg mL–1). Both COC and CFO were pooled in groups (5/group) and tested for presence of BVDV using sensitive real-time PCR method. Only 2.5% of the COC were BVDV positive and all of the CFO were BVDV negative. Additionally, 293 embryos were produced from 14 different cell lines using the previously described SCNT procedure (Zhou et al. 2008 Mol Reprod Dev. 75, 1281–1289). These embryos were generated using in vitro-matured oocytes collected as 23 different batches over 7 months. The embryos were cultured in vitro to blastocyst stage and then tested for BVDV. All of the 293 SCNT embryos (100%) were BVDV negative. In conclusion, under these SCNT embryo production conditions, a small portion of COC were BVDV positive. However, all CFO and SCNT embryos were BVDV negative. Therefore, the risk of disease transmission using abattoir oocytes and SCNT procedure is relatively low and can be eliminated by using a combination of cumulus cell removal and adequate oocyte washing procedures. F. Arenivas, B. Findeisen, V. Farrar, E. Hwang helped with the SCNT embryo production for this study.
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Posner, Mason, Kelly L. Murray, Matthew S. McDonald, Hayden Eighinger, Brandon Andrew, Amy Drossman, Zachary Haley, Justin Nussbaum, Larry L. David, and Kirsten J. Lampi. "The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function." PeerJ 5 (November 27, 2017): e4093. http://dx.doi.org/10.7717/peerj.4093.
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Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4–5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1–6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4–7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.
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Hwang, S. U., J. D. Yoon, K. Y. Eun, H. G. Kim, and S. H. Hyun. "22 PRODUCTION OF PORCINE TRANSGENIC CELL LINE INSERTED WITH SV40LT, EGFRvIII Gene, AND INDUCIBLE CreERT SYSTEM." Reproduction, Fertility and Development 28, no. 2 (2016): 141. http://dx.doi.org/10.1071/rdv28n2ab22.
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Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P < 0.05) and blastocyst formation rates (19.5 ± 1.2 and 5.6 ± 1.8 in the normal and SV40LT group, respectively; P < 0.05) of transgenic SCNT embryos was significantly lower than the case of using normal cells. In addition, we evaluated the transgenic SCNT embryo development of the donor cell transfected with SV40LT and HrasV12 genes (SV40LT+HrasV12 group). As a results, there was no significant difference between the groups in the cleavage rates, but blastocyst formation rates of transfected SCNT embryos (SV40LT+HrasV12 group) was significantly lower than the case of using normal cells (19.5 ± 1.2 and 6.2 ± 1.8 in the normal and SV40LT+HrasV12 group, respectively; P < 0.05). Genes SV40LT or HrasV12 showed a negative effect on SCNT cloned embryo development. Therefore, a Cre/loxP inducible system was applied to producing donor cells transfected with EGFRvIII and SV40LT gene. As a result, the cleavage rates (73.8 ± 4.0 and 68.6 ± 6.6 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) and blastocyst formation rate (19.5 ± 1.2 and 23.0 ± 3.7 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) were improved to the same level, when used as a donor cell to a normal cell. In conclusion, these results indicated that harmful effects of transgenic SCNT embryo development caused by the characteristics of the inserted genes can be overcome through the inducible system. Further studies are needed to experiment with mRNA expression of apoptotic gene and target gene in 4- to 8-cell embryos and blastocysts. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.
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Amali, Aseervatham Anusha, Ravikumar Deepa Rekha, Cliff Ji-Fan Lin, Wei-Lun Wang, Hong-Yi Gong, Gour-Mour Her, and Jen-Leih Wu. "Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis." Journal of Biomedical Science 13, no. 2 (February 3, 2006): 225–32. http://dx.doi.org/10.1007/s11373-005-9055-5.
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Arteaga, Cristina, Nuria Boix, Elisabet Teixido, Fernanda Marizande, Santiago Cadena, and Alberto Bustillos. "The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds." Molecules 26, no. 19 (September 24, 2021): 5786. http://dx.doi.org/10.3390/molecules26195786.
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The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except β-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, β-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.
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Erdenetogtokh, P., S. Ganbat, and Hiroshi Suzuki. "Possibility of elimination of pathogen from Babesia infected animals by means of embryo transfer." Mongolian Journal of Agricultural Sciences 11, no. 2 (November 24, 2014): 36–42. http://dx.doi.org/10.5564/mjas.v11i2.214.
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Babesia infections occur mainly in animals, and are transmitted by ticks. The severity of the diseases varies considerably depending on the species of Babesia involved as well as the immune response of the infected animal. In Mongolia infection produced by Babesia parasites is widely spread, provoking severe damage to the agricultural and economic sectors. Currently, strategies to control and prevent the infection are inefficient. Indeed, the necessity to look for suitable and accessible strategies to obtain animals free from the infection is needed. Currently, assisted reproductive technologies (ART) are used for the improvement of productivity in livestock. Moreover, embryo transfer seams to be useful approach to obtain clean embryos obtained from infected animals. Therefore, by using a mice model (ICR) infected with Babesia microti, an alternative method to obtain animals free from infection was examined. ICR mice at 8 weeks old were challenged with 0.2 ml of 1x107 IRBC/ml by i.p injection. After infection, superovulation was induced and then embryos were obtained and washed. Then, their development stage along with their morphological characteristics were monitored. In vitro embryos obtained from uninfected mice were used as a control group. The results indicate that the infection does not have any influence on pre-implantation embryonic development and morphological characteristics. Thus, we suggest that embryos obtained from infected animals might be useful for embryo transfer in order to improve productivity of livestock and reduce the risk of congenital infection. In summary, ART such as embryo transfer might be an useful technique in countries where Babesiosis is an endemic disease. DOI: http://dx.doi.org/10.5564/mjas.v11i2.214 Mongolian Journal of Agricultural Sciences Vol.11(2) 2013 pp.36-42
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Smith, Tiffany N., and Steven B. Oppenheimer. "Involvement of l(–)-rhamnose in sea urchin gastrulation: a live embryo assay." Zygote 23, no. 2 (October 18, 2013): 222–28. http://dx.doi.org/10.1017/s0967199413000452.
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SummaryThe sea urchin embryo is a National Institutes of Health model system that has provided major developments, and is important in human health and disease. To obtain initial insights to identify glycans that mediate cellular interactions, Lytechinus pictus sea urchin embryos were incubated at 24 or 30 h post-fertilization with 0.0009–0.03 M alpha-cyclodextrin, melibiose, l(–)-rhamnose, trehalose, d(+)-xylose or l(–)-xylose in lower-calcium artificial sea water (pH 8.0, 15°C), which speeds the entry of molecules into the interior of the embryos. While α-cyclodextrin killed the embryos, and l(–)-xylose had small effects at one concentration tested, l(–)-rhamnose caused substantially increased numbers of unattached archenterons and exogastrulated embryos at low glycan concentrations after 18–24 h incubation with the sugar. The results were statistically significant compared with the control embryos in the absence of sugar (P < 0.05). The other sugars (melibiose, trehalose, d(+)-xylose) had no statistically significant effects whatsoever at any of the concentrations tested. In total, in the current study, 39,369 embryos were examined. This study is the first demonstration that uses a live embryo assay for a likely role for l(–)-rhamnose in sea urchin gastrula cellular interactions, which have interested investigators for over a century.
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Mendis, Janthri C., Thejani K. Tennakoon, and Chanika D. Jayasinghe. "Zebrafish Embryo Toxicity of a Binary Mixture of Pyrethroid Insecticides: d-Tetramethrin and Cyphenothrin." Journal of Toxicology 2018 (December 26, 2018): 1–8. http://dx.doi.org/10.1155/2018/4182694.
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Pesguard FG161™, a mixture of d-tetramethrin and cyphenothrin (1:3 ratio), is extensively used to achieve rapid control of adult dengue vector, Aedes aegypti, during the disease outbreaks. Both d-tetramethrin and cyphenothrin are synthetic pyrethroids that are known to have adverse effects on non-mammalian organisms such as fish. The present study intended to use zebrafish embryo toxicity model to investigate the toxic effect of the above binary mixture on fish. Particularly, zebrafish embryo toxicity model provides an alternative to acute fish toxicity tests in terms of animal welfare perspective as the embryos are not considered live until 5 days after fertilization. The zebrafish embryos (2 hrs after fertilization) were exposed to a binary mixture of pyrethroids at different concentrations (d-tetramethrin: 0.01 – 1.20 μmolL-1 and cyphenothrin: 0.03 – 3.20 μmolL-1) for 24, 48, and 72 hrs at room temperature (26°C) according to the OECD guideline no. 236. Percentage mortality of embryos were calculated by observing the lethal endpoints and LC50 values were calculated for each time interval employing the probit analysis. This binary mixture was highly toxic to zebrafish embryos and was found to be concentration and time dependent. LC50 values at 24 hrs (d-tet: 0.58 μmolL-1, cyp: 1.74 μmolL-1) were significantly reduced in 48 hrs (d-tet: 0.11 μmolL-1, cyp: 0.33 μmolL-1) and 72 hrs (d-tet: 0.03 μmolL-1, cyp: 0.09 μmolL-1). Coagulation of embryos was the most common lethal effect observed and lack of somite formation and lack of heartbeat were also observed. The present study revealed that the binary mixture is highly toxic to zebrafish embryos even when based on nominal concentrations. Hence, extensive use of these pesticides could be detrimental to fish population and integrated vector control methods which involve the minimum use of insecticides are recommended. Further, this study highlights the applicability of zebrafish embryo toxicity model as an alternative method to investigate the toxicity of pyrethroids to fish.
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Sang, Helen. "DISEASE RESISTANCE AND OTHER APPLICATIONS OF TRANSGENESIS IN THE CHICKEN." Reproduction, Fertility and Development 25, no. 1 (2013): 320. http://dx.doi.org/10.1071/rdv25n1ab345.
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Genetic modification of the chicken in terms of gene addition is now robust and efficient. Transgenes can be introduced by injection of lentiviral vectors into chick embryos or by transfection of transposon vectors into embryos or primordial germ cells in vitro. Lentiviral vectors are limited in the size of transgene they can incorporate but we have generated several different transgenic lines using HIV-derived vectors and have observed high levels of transgene expression and tissue-specific expression using regulatory sequences from several genes. M. McGrew (The Roslin Institute) has established primordial germ cell lines and effective methods for transfection with piggyBac and Tol2 transposon vectors. The primordial cells are injected into chick embryos where they populate the developing gonads and contribute to the germline in mature birds. The availability of primordial germ cell lines will also form the basis of using artificial site-specific nucleases for gene knockout and potentially gene targeting in the chicken. These technologies facilitate the application of transgenesis in the chicken for basic research and for potential applications in poultry breeding. The chick embryo is an invaluable model for studying vertebrate development as the embryos can be accessed in ovo or in culture at the earlier stages of development. Embryos can be transfected with transgenes by electroporation and manipulated to study many aspects of development. We are developing transgenic chickens in which fluorescent protein reporters are expressed either ubiquitously or in targeted cell types. These form the basis of novel tools for increasing the value of the chick embryo in studying development. We provide fertile eggs from these lines to other research groups and are investigating the development of macrophages using a macrophage-targeted reporter. The potential for the use of genetic modification to be used in poultry breeding can now be explored. Commercial poultry production is challenged by several major pathogens including avian influenza. Flocks can be protected by good biosecurity measures and/or vaccination but vaccination is not always effective. It may be possible to add novel genes to the chicken genome targeting avian influenza virus replication. We are developing this approach (with L. Tiley, Cambridge University) and have generated transgenic chickens that do not transmit avian influenza when directly infected with H5N1 virus.
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Deutsch, Varda, Yona Farnoushi, Michal Cipok, Sigi Kay, Elizabeth Naparstek, and Ronald Goldstein. "A New Xenograft Model for Studying Multiple Myeloma and Drug Response." Blood 116, no. 21 (November 19, 2010): 4073. http://dx.doi.org/10.1182/blood.v116.21.4073.4073.
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Abstract Abstract 4073 While new treatment options are available, multiple myeloma (MM) still remains an incurable malignancy of plasma cells with a grim prognosis. Practical in vivo models to study human MM may enable a better understanding of the biology of the disease, and better optimization of therapeutic strategies. The best current xenograft model, the immune-deficient NOD/SCID mice, recapitulates MM in vivo, however, the price is very costly and maintenance complex, with >1 month required to establish engraftment. Our goal was to develop a user friendly rapid alternative xenograft system for the preclinical assessment of MM growth and therapy. We recently described this new in-vivo system for studying human leukemia in the pre-immune turkey embryo 1,2. These embryos are inexpensive, require no maintenance, and are easily manipulated experimentally. Described here are the first attempts at application of this novel system to study MM and test therapies. Cell lines ARH-77 and CAG line and fresh patient cells (5 × 106/embryo) were injected IV into turkey egg chorioallantoic membrane veins on embryonic day E11. Engraftment of human cells in hematopoietic organs, bone marrow (BM) and liver was detected 7 days later (E18) by RTPCR, immunohistochemistry and flow cytometry and by circulating free light chain (6-25 mg/L) in the peripheral blood of 100% of the injected cell lines and 50% of patients myelomas. Treatment with Velcade (Bortezomib) or Revlimid IV on E13 (48 hours after MM cell injection), at drug levels that were precalibrated to be non-toxic to the developing embryonic BM, dramatically reduced engraftment, demonstrating the utility of this new model for testing drug activity in vivo. ARH-77 cells, detected by flow cytometry of the embryonic BM cells with anti-human CD19, CD38 and CD138, were inhibited from 8.5% engraftment to 0.72% after a single Velcade treatment, with an 18 fold decrease compared to untreated embryos in the ratio of human to avian cells in BM tissue. determined by Q-RT-PCR analysis of human alpha satellite and avian GAPDH DNA normalized per cell. Very similar results were obtained with Revlimid. The results presented suggest that with further work the turkey embryo model may provide an affordable, rapid and practical xenograft system in vivo for studying the biology of MM, for affordably testing MM therapies, as well for developing a new method for individualized patient screening for response or resistance to particular therapeutic agents. 1. Taizi M, Deutsch VR, Leitner A, Ohana A, Goldstein RS. A novel and rapid in vivo system for testing therapeutics on human leukemias. Exp Hematol. 2006;34:1698-1708. 2. Grinberg I, Reis A, Ohana A, et al. Engraftment of human blood malignancies to the turkey embryo: a robust new in vivo model. Leuk Res. 2009;33:1417-1426. Disclosures: No relevant conflicts of interest to declare.
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Bazzano, María Victoria, Gisela Belén Sarrible, Martín Berón de Astrada, and Evelin Elia. "Obesity modifies the implantation window and disrupts intrauterine embryo positioning in rats." Reproduction 162, no. 1 (July 1, 2021): 61–72. http://dx.doi.org/10.1530/rep-21-0015.
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Obesity is a chronic disease that impairs female reproduction. When gestation is achieved, maternal obesity can cause offspring’s health complications. We intended to evaluate the effects of maternal pre-conceptional obesity on uterine contractile activity, embryo implantation and offspring development. Using cafeteria diet-induced obesity as an animal model, we found that maternal obesity delays embryo transport from the oviduct to the uterus and alters the intrauterine embryo positioning. Adrenergic receptor (AR) signaling is involved in embryo positioning, so all AR isoforms were screened in the pre-implantation uteri. We found that the β2AR is the dominant isoform in the rat uteri and that obesity causes its upregulation. Although β2AR activation is known to induce uterine relaxation, higher spontaneous contractile activity was detected in obese dams. Uteri from obese dams showed a higher sensitivity to salbutamol (a selective agonist of β2AR) than controls, consistent with the higher β2AR levels detected in those animals. Despite this, in obese dams, some embryos were still in the oviduct at the predicted time of initial embryo attachment, embryo implantation is successfully carried out since the total number of fetuses on gd 18.5 were similar between control and obese dams. These findings show that obesity is modifying the implantation window. Moreover, we found that maternal obesity resulted in macrosomia in the offspring, which is an important predictor of fetal programming of postnatal health. Hence, our results show that maternal obesity prior to pregnancy not only disturbs the implantation process, but also affects offspring development.
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Marin, Clara, Ximo Garcia-Dominguez, Laura Montoro-Dasi, Laura Lorenzo-Rebenaque, José S. Vicente, and Francisco Marco-Jimenez. "Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks." Animals 10, no. 4 (April 1, 2020): 598. http://dx.doi.org/10.3390/ani10040598.
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In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.
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Kunkanjanawan, Tanut, Parinya Noisa, and Rangsun Parnpai. "Modeling Neurological Disorders by Human Induced Pluripotent Stem Cells." Journal of Biomedicine and Biotechnology 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/350131.
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Studies of human brain development are critical as research on neurological disorders have been progressively advanced. However, understanding the process of neurogenesis through analysis of the early embryo is complicated and limited by a number of factors, including the complexity of the embryos, availability, and ethical constrains. The emerging of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) has shed light of a new approach to study both early development and disease pathology. The cells behave as precursors of all embryonic lineages; thus, they allow tracing the history from the root to individual branches of the cell lineage tree. Systems for neural differentiation of hESCs and iPSCs have provided an experimental model that can be used to augmentin vitrostudies ofin vivobrain development. Interestingly, iPSCs derived from patients, containing donor genetic background, have offered a breakthrough approach to study human genetics of neurodegenerative diseases. This paper summarizes the recent reports of the development of iPSCs from patients who suffer from neurological diseases and evaluates the feasibility of iPSCs as a disease model. The benefits and obstacles of iPSC technology are highlighted in order to raising the cautions of misinterpretation prior to further clinical translations.
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Kaneko, T., and T. Mashimo. "222 PRODUCTION OF KNOCKOUT RATS BY USING ZINC FINGER NUCLEASES AND TAL EFFECTOR NUCLEASES." Reproduction, Fertility and Development 26, no. 1 (2014): 225. http://dx.doi.org/10.1071/rdv26n1ab222.
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The rat has been used as an important animal for understanding human diseases. Genetically engineered rat strains are used as a human disease model in various research fields. Genetically engineered rat strains are now being routinely produced, not only as transgenic animals but also using gene knockout techniques. Recently, zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN) have enabled editing targeted genes without using embryonic stem cells. These techniques have been applied for production of the knockout and knockin animals. We here studied that the effects of gene targeting by ZFN and TALEN introduced into rat embryos for efficient production of knockout rats. We custom-designed ZFN and TALEN plasmids targeted rat interleukin 2 receptor gamma (Il2rg) gene. Each mRNA was transcribed in vitro from these plasmids. Final concentration of mRNA was adjusted at 10 ng μL–1 in sterilized water for microinjection. Messenger RNA was injected into rat pronuclear stage embryos. The embryos were then cultured in vitro to the 2-cell stage, and were transferred into oviducts of pseudopregnant females. The rate of development of offspring of embryos and effects of editing targeted genes were examined. Of 41 two-cell embryos introduced ZFN after embryo transfer, 9 embryos (22%) developed to offspring. Three offspring (33%) had an edited targeted gene locus. In the embryos introduced TALEN, 30% (6 offspring) of embryos developed to offspring after embryo transfer and all offspring had an edited targeted gene locus. This study demonstrated that the ZFN and TALEN mRNA was active after introduction into rat embryos. Knockout rats could be produced by introduction of ZFN and TALEN into rat embryos. ZFN and TALEN will provide a powerful new approach for targeted gene editing not only in rats but also in other animal species.
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Fleming, T. P., M. A. Velazquez, and J. J. Eckert. "Embryos, DOHaD and David Barker." Journal of Developmental Origins of Health and Disease 6, no. 5 (May 8, 2015): 377–83. http://dx.doi.org/10.1017/s2040174415001105.
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The early embryo and periconceptional period is a window during which environmental factors may cause permanent change in the pattern and characteristics of development leading to risk of adult onset disease. This has now been demonstrated across small and large animal models and also in the human. Most evidence of periconceptional ‘programming’ has emerged from maternal nutritional models but also other in vivo and in vitro conditions including assisted reproductive treatments, show consistent outcomes. This short review first reports on the range of environmental in vivo and in vitro periconceptional models and resulting long-term outcomes. Second, it uses the rodent maternal low protein diet model restricted to the preimplantation period and considers the stepwise maternal-embryonic dialogue that comprises the induction of programming. This dialogue leads to cellular and epigenetic responses by the embryo, mainly identified in the extra-embryonic cell lineages, and underpins an apparently permanent change in the growth trajectory during pregnancy and associates with increased cardiometabolic and behavioural disease in adulthood. We recognize the important advice of David Barker some years ago to investigate the sensitivity of the early embryo to developmental programming, an insight for which we are grateful.
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Jevtic, Predrag, and Daniel Levy. "Elucidating nuclear size control in the Xenopus model system." Veterinarski glasnik 72, no. 1 (2018): 1–13. http://dx.doi.org/10.2298/vetgl170731012j.
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Background. Nuclear size is a tightly regulated cellular feature. Mechanisms that regulate nuclear size and the functional significance of this regulation are largely unknown. Nuclear size and morphology are often altered in many diseases, such as cancer. Therefore, understanding the mechanisms that regulate nuclear size is crucial to provide insight into the role of nuclear size in disease. Scope and Approach. The goal of this review is to summarize the most recent studies about the mechanisms and functional significance of nuclear size control using the Xenopus model system. First, this review describes how Xenopus egg extracts, embryos, and embryo extracts are prepared and used in scientific research. Next, the review focuses on the mechanisms and functional effects of proper nuclear size control that have been learned using the Xenopus system. Key Findings and Conclusions. Xenopus is an excellent in vivo and in vitro experimental platform to study mechanisms of nuclear size control. Given its close evolutionary relationship with mammals and that most cellular processes and pathways are highly conserved between Xenopus and humans, the Xenopus system has been a valuable tool to advance biomedical research. Some of the mechanisms that regulate nuclear size include components of nuclear import such as importin ? and NTF2, nuclear lamins, nucleoporins, proteins that regulate the morphology of the endoplasmic reticulum, and cytoskeletal elements.
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Fleming, Tom P., Adam J. Watkins, Congshan Sun, Miguel A. Velazquez, Neil R. Smyth, and Judith J. Eckert. "Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo’s potential, affecting adult health." Reproduction, Fertility and Development 27, no. 4 (2015): 684. http://dx.doi.org/10.1071/rd14455.
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Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and ‘decisions’ made by embryos to optimise their own development, but with lasting consequences.
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Martins, D. S., N. Z. Saraiva, J. M. Garcia, C. E. Ambrósio, M. Zatz, and M. A. Miglino. "201 CHARACTERIZATION OF CANINE EMBRYONIC GERM CELLS: AN EXPERIMENTAL MODEL FOR CELL THERAPY." Reproduction, Fertility and Development 18, no. 2 (2006): 209. http://dx.doi.org/10.1071/rdv18n2ab201.
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Dogs represent excellent models to test different approaches before use for human therapy. Studies with animal models have suggested that the transplant of stem cells may have success in the treatment of degenerative diseases such as Parkinson's disease, diabetes, Duchenne muscular dystrophy (DMD), and acquired lesions. Embryonic stem cells are pluripotent and therefore have the potential to form all tissues. Our research aims to contribute to the treatment of DMD through the isolation and identification of embryonic germ cells and the development of the methodology of cellular differentiation for future transplantation into dystrophic dogs. Mongrel female dogs were ovariehysterectomized between 25 and 30 days of pregnancy. For recovery of embryos, the excised uterine horns were flushed with heparinized PBS. Samples collected from somites near the mesonephros area of four embryos recovered at 22 to 24 days of pregnancy and designated as A1 through A4 were dissociated and placed in culture. Isolated embryonic cells were allowed to plate onto monolayers of canine fibroblast cells. Flow cytometry was used to identify CD34+ markers. Isolated compact colonies of embryonic germ cells were seen growing around tissue fragments at 7 days of culture and remained in the undifferentiated stage until approximately 21 days in culture. At 14 days of explant, cell colonies were analyzed by flow cytometry. Cells from the A2 embryos contained the highest number of CD34+ cells, whereas no cells from A4 embryos showed specificity for the marker. A small proportion (2.12%) of cells from embryos A1 and A3 showed specificity for the CD34 marker. A quantity of A2 embryo cells that had maintained stem cell characteristics were frozen for future studies. Our results suggest that although spontaneous differentiation occurred, a small population of cells maintain the characteristics of stem cells. We are currently trying to improve the methodology to maintain cells undifferentiated for longer periods and to better control the specific differentiation process.
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Benyumov, Alexey O., Polla Hergert, Jeremy Herrera, Mark Peterson, Craig Henke, and Peter B. Bitterman. "A Novel Zebrafish Embryo Xenotransplantation Model to Study Primary Human Fibroblast Motility in Health and Disease." Zebrafish 9, no. 1 (March 2012): 38–43. http://dx.doi.org/10.1089/zeb.2011.0705.
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Salinas, C. E., C. E. Blanco, M. Villena, E. J. Camm, J. D. Tuckett, R. A. Weerakkody, A. D. Kane, et al. "Cardiac and vascular disease prior to hatching in chick embryos incubated at high altitude." Journal of Developmental Origins of Health and Disease 1, no. 1 (October 1, 2009): 60–66. http://dx.doi.org/10.1017/s2040174409990043.
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The partial contributions of reductions in fetal nutrition and oxygenation to slow fetal growth and a developmental origin of cardiovascular disease remain unclear. By combining high altitude with the chick embryo model, we have previously isolated the direct effects of high-altitude hypoxia on growth. This study isolated the direct effects of high-altitude hypoxia on cardiovascular development. Fertilized eggs from sea-level or high-altitude hens were incubated at sea level or high altitude. Fertilized eggs from sea-level hens were also incubated at high altitude with oxygen supplementation. High altitude promoted embryonic growth restriction, cardiomegaly and aortic wall thickening, effects which could be prevented by incubating eggs from high-altitude hens at sea level or by incubating eggs from sea-level hens at high altitude with oxygen supplementation. Embryos from high-altitude hens showed reduced effects of altitude incubation on growth restriction but not on cardiovascular remodeling. The data show that: (1) high-altitude hypoxia promotes embryonic cardiac and vascular disease already evident prior to hatching and that this is associated with growth restriction; (2) the effects can be prevented by increased oxygenation; and (3) the effects are different in embryos from sea-level or high-altitude hens.
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Aleksanyan, Heghush, Jing Liang, Stan Metzenberg, and Steven B. Oppenheimer. "Terminal alpha-d-mannosides are critical during sea urchin gastrulation." Zygote 24, no. 5 (May 18, 2016): 775–82. http://dx.doi.org/10.1017/s0967199416000113.
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SummaryThe sea urchin embryo is a United States National Institutes of Health (NIH) designated model system to study mechanisms that may be involved in human health and disease. In order to examine the importance of high-mannose glycans and polysaccharides in gastrulation, Lytechinus pictus embryos were incubated with Jack bean α-mannosidase (EC 3.2.1.24), an enzyme that cleaves terminal mannose residues that have α1–2-, α1–3-, or α1–6-glycosidic linkages. The enzyme treatment caused a variety of morphological deformations in living embryos, even with α-mannosidase activities as low as 0.06 U/ml. Additionally, formaldehyde-fixed, 48-hour-old L. pictus embryos were microdissected and it was demonstrated that the adhesion of the tip of the archenteron to the roof of the blastocoel in vitro is abrogated by treatment with α-mannosidase. These results suggest that terminal mannose residues are involved in the adhesion between the archenteron and blastocoel roof, perhaps through a lectin-like activity that is not sensitive to fixation.
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Mo, Yun Jeong, Yu-Seon Kim, Minseok S. Kim, and Yun-Il Lee. "Advantages of Adult Mouse Dorsal Root Ganglia Explant Culture in Investigating Myelination in an Inherited Neuropathic Mice Model." Methods and Protocols 5, no. 4 (July 22, 2022): 66. http://dx.doi.org/10.3390/mps5040066.
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A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease.
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Tonon, Federica, Stefano Di Bella, Gabriele Grassi, Roberto Luzzati, Paolo Ascenzi, Alessandra di Masi, and Cristina Zennaro. "Extra-Intestinal Effects of C. difficile Toxin A and B: An In Vivo Study Using the Zebrafish Embryo Model." Cells 9, no. 12 (December 1, 2020): 2575. http://dx.doi.org/10.3390/cells9122575.
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C.difficile infection (CDI) is not a merely “gut-confined” disease as toxemia could drive the development of CDI-related extra-intestinal effects. These effects could explain the high CDI-associated mortality, not just justified by diarrhea and dehydration. Here, the extra-intestinal effects of toxin A (TcdA) and B (TcdB) produced by C. difficile have been studied in vivo using the zebrafish embryo model. Noteworthy, protective properties of human serum albumin (HSA) towards toxins-induced extra-intestinal effects were also addressed. Zebrafish embryos were treated with TcdA, TcdB and/or HSA at 24 h post-fertilization. Embryos were analyzed for 48 h after treatment to check vital signs and morphological changes. Markers related to cardio-vascular damage and inflammation were evaluated by Real-Time quantitative PCR and/or western blotting. Both toxins induced cardiovascular damage in zebrafish embryos by different mechanisms: (i) direct toxicity (i.e., pericardial edema, cardiac chambers enlargement, endothelial alteration); (ii) increased hormonal production and release (i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), (iii) alteration of the vascular system through the increase of the vascular endothelial growth factor (VEGF-A) levels, as well as of its receptors, (iv) pro-inflammatory response through high cytokines production (i.e., CXCL8, IL1B, IL6 and TNFα) and (v) cell-mediated damage due to the increase in neutrophils number. In addition to cardiovascular damage, we observe skin alteration and inflammation. Finally, our data indicate a protective effect of HSA toward the toxins induced extra-intestinal effects. Together, our findings can serve as a starting point for humans’ studies to substantiate and understand the extra-intestinal effects observed in CDI patients.
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Nasser, L. F., S. C. Feliú, E. Rodríguez, K. Mojica, E. G. Oliveira, A. C. Basso, J. H. F. Pontes, A. Nagele, R. A. C. Rabel, and M. B. Wheeler. "100 COMPARISON OF PREGNANCY RATES OF FRESH IN VITRO-PRODUCED BOS INDICUS EMBRYOS PRODUCED IN THE SAME LABORATORY BUT COLLECTED AND TRANSFERRED IN PANAMA OR THE DOMINICAN REPUBLIC." Reproduction, Fertility and Development 26, no. 1 (2014): 164. http://dx.doi.org/10.1071/rdv26n1ab100.
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Because of Panama's stricter sanitary status, a specialised protocol was developed with the Department of Agriculture in the Dominican Republic to legalize the exchange of biological materials (oocytes/embryos). This protocol allows the team of specialised technicians, currently working in Born® Animal Biotechnology's Panamanian facility, to operate using the same in vitro bovine embryo production system (IVP, In vitro Brasil®) to service Dominican producers. Because the donors are not located at a specific centre with controlled sanitary management, a special protocol was developed in which blood tests were done to certify that the entirety of the herd at each client's farm was free of infectious bovine rhinotracheitis, DBVD, leptospirosis, leucosis, brucellosis, and tuberculosis. As timing during IVP is an essential variable that can have detrimental effects on the final results, precautions were taken to ensure that the oocytes arrived at the Panamanian laboratory facility within 24 h of aspiration. A portable incubator was used to transport oocytes and embryos during the import and export portions of the procedure. A comparison of pregnancy rates based on oocyte source and recipient transfers from September 2012 until May 2013 was analysed with ?2 (Table 1). The number of embryos produced in Panama was significantly higher than in the Dominican Republic, which was likely due to the larger number of donors and oocytes from the Panama herd. However, pregnancy rate was higher in the Dominican Republic likely because of the health status of the Dominican recipients, which were free of the diseases mentioned above. Recipients were the same type and breed and under similar management conditions in both countries. The disease status aspect will be examined with greater numbers of animals in the future. The data suggest that the present IVP and recipient management protocols could serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.In vitro embryo production and pregnancy rates of Bos indicus embryos transferred in Panama or the Dominican Republic (September 2012 through May 2013)
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Twagirumukiza, Gratien, and Edouard Singirankabo. "Mathematical analysis of a delayed HIV/AIDS model with treatment and vertical transmission." Open Journal of Mathematical Sciences 5, no. 1 (March 22, 2021): 128–46. http://dx.doi.org/10.30538/oms2021.0151.
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None can underestimate the importance of mathematical modelling for their role in clarifying dynamics of epidemic diseases. They can project the progress of the disease and demonstrate the result of the epidemic to public health in order to take precautions. HIV attracts global attention due to rising death rates and economic burdens and many other consequences that it leaves behind. Up to date, there is no medicine and vaccine of HIV/AIDS but still many researches are conducted in order to see how to mitigate this epidemic and reduce the death rate or increase the life expectancy of those who are infected. A delayed HIV/AIDS treatment and vertical transmission model has been investigated. The model took into account both infected people from the symptomatics group and asymptomatic group to join AIDS group. We considered that a child can be infected from the mother to an embryo, fetus or childbirth. Those who are infected, it will take them some time to get mature and spread the disease. By using mathematical model, reproduction number, positivity, boundedness, and stability analysis were determined. The results showed that the model is much productive if time delay is considered.
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Nóbrega, Ana, Ana C. Maia-Fernandes, and Raquel P. Andrade. "Altered Cogs of the Clock: Insights into the Embryonic Etiology of Spondylocostal Dysostosis." Journal of Developmental Biology 9, no. 1 (January 29, 2021): 5. http://dx.doi.org/10.3390/jdb9010005.
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Spondylocostal dysostosis (SCDO) is a rare heritable congenital condition, characterized by multiple severe malformations of the vertebrae and ribs. Great advances were made in the last decades at the clinical level, by identifying the genetic mutations underlying the different forms of the disease. These were matched by extraordinary findings in the Developmental Biology field, which elucidated the cellular and molecular mechanisms involved in embryo body segmentation into the precursors of the axial skeleton. Of particular relevance was the discovery of the somitogenesis molecular clock that controls the progression of somite boundary formation over time. An overview of these concepts is presented, including the evidence obtained from animal models on the embryonic origins of the mutant-dependent disease. Evidence of an environmental contribution to the severity of the disease is discussed. Finally, a brief reference is made to emerging in vitro models of human somitogenesis which are being employed to model the molecular and cellular events occurring in SCDO. These represent great promise for understanding this and other human diseases and for the development of more efficient therapeutic approaches.
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Jeon, Y., Y. K. Kim, J. D. Yoon, L. Cai, S. U. Hwang, E. Kim, S. Lee, E. B. Jeung, and S. H. Hyun. "4 PRODUCTION OF 11β-HYDROXYSTEROID DEHYDROGENASE TYPE 1 (11β-HSD1) OVER-EXPRESSED PIGS FOR THE STUDY OF METABOLIC SYNDROME DISEASE." Reproduction, Fertility and Development 26, no. 1 (2014): 116. http://dx.doi.org/10.1071/rdv26n1ab4.
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Metabolic syndrome shows a complication at risk for cardiovascular disease and diabetes. With the high prevalence of obesity globally, the incidence of metabolic syndrome is increased. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, we attempted to develop large-animal model for the study of metabolic syndrome disease. Some studies have shown that constitutive overexpression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome. Therefore, we tried to produce the 11β-HSD1 gene overexpressed pig by somatic cell nuclear transfer (SCNT). First, we established 11β-HSD1 overexpressed cells for the preparation of the transgenic (TG) donor cells. Porcine fetal fibroblasts were transfected with cytomegalovirus vector that included the porcine 11β-HSD1 gene. The 11β-HSD1-TG cell was injected into the enucleated ooplasm to produce 11β-HSD1-TG cloned embryos. In total, 833 TG porcine SCNT embryos (TG-SCNT embryos) were made. Of these, 155 TG-SCNT embryos were cultured in procine zygote medium-3 to evaluate the in vitro developmental potential of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 109 embryos (70.3%) were cleaved at 48 h. On Day 7, 31 transgenic porcine SCNT embryos (20.0%) developed to the blastocyst stage. The remaining 678 TG-SCNT embryos were transferred to 6 surrogates (average 113 embryos per surrogate). On 25 days after embryo transfer, 2 surrogates were diagnosed as pregnant (pregnancy rate, 33.3%). On Day 114, we obtained 9 live piglets and 3 stillborn piglets from 2 surrogates. By PCR analysis, we confirmed that 1 live piglet and 2 stillborn piglets were integrated with 11β-HSD1 gene. We succeeded to obtain TG piglets at sixth trials, and for the re-cloning by SCNT, a stable cell line transfected with the 11β-HSD1 gene was established from a TG cloned piglet. This study presents new possibilities for large-animal model development for the study of metabolic syndrome disease. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
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Harrington, Jamie K., Robert Sorabella, Abigail Tercek, Joseph R. Isler, and Kimara L. Targoff. "Nkx2.5 is essential to establish normal heart rate variability in the zebrafish embryo." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 313, no. 3 (September 1, 2017): R265—R271. http://dx.doi.org/10.1152/ajpregu.00223.2016.
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Heart rate variability (HRV) has become an important clinical marker of cardiovascular health and a research measure for the study of the cardiac conduction system and its autonomic controls. While the zebrafish ( Danio rerio) is an ideal vertebrate model for understanding heart development, HRV has only recently been investigated in this system. We have previously demonstrated that nkx2.5 and nkx2.7, two homologues of Nkx2–5 expressed in zebrafish cardiomyocytes, play vital roles in maintaining cardiac chamber-specific characteristics. Given observed defects in ventricular and atrial chamber identities in nkx2.5−/− embryos coupled with conduction system abnormalities in murine models of Nkx2.5 insufficiency, we postulated that reduced HRV would serve as a marker of poor cardiac health in nkx2.5 mutants and in other zebrafish models of human congenital heart disease. Using live video image acquisition, we derived beat-to-beat intervals to compare HRV in wild-type and nkx2.5−/− embryos. Our data illustrate that the nkx2.5 loss-of-function model exhibits increased heart rate and decreased HRV when compared with wild type during embryogenesis. These findings validate HRV analysis as a useful quantitative tool for assessment of cardiac health in zebrafish and underscore the importance of nkx2.5 in maintaining normal heart rate and HRV during early conduction system development.
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Sokolenko, Ekaterina A., Utta Berchner-Pfannschmidt, Saskia C. Ting, Kurt W. Schmid, Nikolaos E. Bechrakis, Berthold Seitz, Theodora Tsimpaki, Miriam Monika Kraemer, and Miltiadis Fiorentzis. "Optimisation of the Chicken Chorioallantoic Membrane Assay in Uveal Melanoma Research." Pharmaceutics 14, no. 1 (December 22, 2021): 13. http://dx.doi.org/10.3390/pharmaceutics14010013.
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The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments.
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Nowik, N., P. Podlasz, A. Jakimiuk, N. Kasica, W. Sienkiewicz, and J. Kaleczyc. "Zebrafish: an animal model for research in veterinary medicine." Polish Journal of Veterinary Sciences 18, no. 3 (September 1, 2015): 663–74. http://dx.doi.org/10.1515/pjvs-2015-0086.
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Abstract The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic disorders including fatty liver disease and diabetes. The zebrafish is also a valuable tool as a model in behavioral studies connected with feeding, predator evasion, habituation and memory or lateralized control of behavior. The aim of the present article is to familiarize the reader with the possibilities of Danio rerio as an experimental model for veterinary medicine.
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Kasza, Karen E., Sara Supriyatno, and Jennifer A. Zallen. "Cellular defects resulting from disease-related myosin II mutations in Drosophila." Proceedings of the National Academy of Sciences 116, no. 44 (October 15, 2019): 22205–11. http://dx.doi.org/10.1073/pnas.1909227116.
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The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.
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Uloza, Virgilijus, Alina Kuzminienė, Sonata Šalomskaitė-Davalgienė, Jolita Palubinskienė, Ingrida Balnytė, Ingrida Ulozienė, Viktoras Šaferis, and Angelija Valančiūtė. "Effect of Laryngeal Squamous Cell Carcinoma Tissue Implantation on the Chick Embryo Chorioallantoic Membrane: Morphometric Measurements and Vascularity." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/629754.
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Background. The aim of this study was to develop chick embryo chorioallantoic membrane (CAM) model of laryngeal squamous cell carcinoma (LSCC) and to evaluate the morphological and morphometric characteristics and angiogenic features of it.Methods. Fresh LSCC tissue samples obtained from 6 patients were implanted onto 15 chick embryo CAMs. Morphological, morphometric, and angiogenic changes in the CAM and chorionic epithelium were evaluated up to 4 days after the tumor implantation. Immunohistochemical analysis (34βE12, CD31, and Ki67 staining) was performed to detect cytokeratins and tumor endothelial cells and to evaluate the proliferative capacity of the tumor before and after implantation on the CAM.Results. The implanted LSCC tissue samples survived on the CAM in all the experiments and retained the essential morphologic characteristics and proliferative capacity of the original tumor. Implants induced thickening of both the CAM (103–417%,p=0.0001) and the chorionic epithelium (70–140%,p=0.0001) and increase in number of blood vessels (75–148%,p=0.0001) in the CAM.Conclusions. This study clarifies that chick embryo CAM is a relevant assay for implanting LSCC tissue and provides the first morphological and morphometric characterization of the LSCC CAM model that opens new perspectives to study this disease.
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Yang, S. Y., P. C. Cheng, and A. W. S. Chan. "6 LENTIVIRAL TRANSGENESIS IN MICE AND NONHUMAN PRIMATES." Reproduction, Fertility and Development 20, no. 1 (2008): 83. http://dx.doi.org/10.1071/rdv20n1ab6.
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Transgenic technology is a powerful tool for investigating gene function and regulation in species ranging from fly to higher primates. The role of transgenic animal modeling has become more prominent in biomedical research; therefore, a highly efficient method for producing transgenic animals is critical for the advancement of animal modeling of genetic disorders, especially in species with limited access such as nonhuman primates. Lentiviral transgenesis is one of most efficient methods in generating transgenic animals, and has been applied in different species including mice, rats, pigs, and cattle. Here we evaluated lentiviral transgenesis by an in-depth investigation on the effect of gene construct and the method of viral delivery in mice; thus the prospect of creating transgenic nonhuman primates can be assessed. Lentiviruses carrying 25 different gene constructs in the same viral backbone were created and microinjected into the cytoplasm or the perivitelline space (PVS) of mouse zygotes; these zygotes were then compared to those subjected to the traditional pronuclear injection (PI) method. Embryo development was not affected by PVS, whereas intracytoplasmic injection produced a mild effect on embryo development, which was dependent on the manipulation skill. We found that intracytoplasmic injection of lentivirus had the highest transgenic rate (weaned pups) of approximately 54.22% (199/367), whereas the transgenic rate using PVS injection was 40.74% (22/54). However, the transgenic rate of PI was only 9.09% (4/44), which was significantly lower than the other two methods. Germline transmission was confirmed in over 90% of the transgenic lines produced by lentiviral gene transfer. In addition to the effect due to gene delivery method, variations in gene transfer efficiency were also observed when lentiviruses with different constructs were used. Our interest was to translate the lentiviral gene transfer technique into nonhuman primates for the development of a model for human disease. We evaluated the in vitro developmental rate of Rhesus macaques embryos that were microinjected into the PVS with lentiviruses carrying mutant genes leading to neurodegenerative diseases. The blastocyst rate of the lentivirus injection group (26%; 62/238) was not different from that of the control (25%; 13/52), which was without lentivirus injection. This indicates the feasibility of applying the lentiviral gene transfer technique to nonhuman primates. We carried out embryo transfers to surrogate female monkeys; however, the confirmation of pregnancy and the success of a developing nonhuman primate model of human disease were not available at the time of this writing. Here we demonstrate that lentiviral transgensis by cytoplasmic injection or PVS injection is a promising method to generate transgenic animals at high efficiency, and is superior to the traditional methods. Thus the production of a nonhuman primate model of human genetic diseases is foreseeable, and will have a significant impact on transgenic animal modeling as well as the advancement of biomedicine. This work was supported by the NCRR/NIH.
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Deutsch, Varda R., Sigi Kay, Yona Farnoushi, Erez Matalon, Tal Ohayon, Shoshana Baron, Hila Jan, Elizabeth Naparstek, and Michal Cipok. "New Xenograft Method For Studying Leukemia and Drug Response In Turkey Embryos." Blood 122, no. 21 (November 15, 2013): 3973. http://dx.doi.org/10.1182/blood.v122.21.3973.3973.
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Abstract Background A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy. Methods BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments. Conclusions The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents. Disclosures: No relevant conflicts of interest to declare.
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Barnett, Sarah E., Anne Herrmann, Liam Shaw, Elisabeth N. Gash, Harish Poptani, Joseph J. Sacco, and Judy M. Coulson. "The Chick Embryo Xenograft Model for Malignant Pleural Mesothelioma: A Cost and Time Efficient 3Rs Model for Drug Target Evaluation." Cancers 14, no. 23 (November 26, 2022): 5836. http://dx.doi.org/10.3390/cancers14235836.
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Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.
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Lee, Banseok, Byoungyun Choi, Youngjae Park, Seokhui Jang, Chunyu Yuan, Chaejin Lim, Jang Ho Lee, Gyun Jee Song, and Kyoung Sang Cho. "Roles of ZnT86D in Neurodevelopment and Pathogenesis of Alzheimer Disease in a Drosophila melanogaster Model." International Journal of Molecular Sciences 23, no. 19 (October 5, 2022): 11832. http://dx.doi.org/10.3390/ijms231911832.
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Zinc is a fundamental trace element essential for numerous biological processes, and zinc homeostasis is regulated by the Zrt-/Irt-like protein (ZIP) and zinc transporter (ZnT) families. ZnT7 is mainly localized in the Golgi apparatus and endoplasmic reticulum (ER) and transports zinc into these organelles. Although previous studies have reported the role of zinc in animal physiology, little is known about the importance of zinc in the Golgi apparatus and ER in animal development and neurodegenerative diseases. In this study, we demonstrated that ZnT86D, a Drosophila ortholog of ZnT7, plays a pivotal role in the neurodevelopment and pathogenesis of Alzheimer disease (AD). When ZnT86D was silenced in neurons, the embryo-to-adult survival rate, locomotor activity, and lifespan were dramatically reduced. The toxic phenotypes were accompanied by abnormal neurogenesis and neuronal cell death. Furthermore, knockdown of ZnT86D in the neurons of a Drosophila AD model increased apoptosis and exacerbated neurodegeneration without significant changes in the deposition of amyloid beta plaques and susceptibility to oxidative stress. Taken together, our results suggest that an appropriate distribution of zinc in the Golgi apparatus and ER is important for neuronal development and neuroprotection and that ZnT7 is a potential protective factor against AD.
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Asad, Zainab, and Chetana Sachidanandan. "Chemical screens in a zebrafish model of CHARGE syndrome identifies small molecules that ameliorate disease-like phenotypes in embryo." European Journal of Medical Genetics 63, no. 2 (February 2020): 103661. http://dx.doi.org/10.1016/j.ejmg.2019.04.018.
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Hakam, Miya Soukaina, Jose Maria Miranda-Sayago, Soren Hayrabedyan, Krassimira Todorova, Patrick Simon Spencer, Asma Jabeen, Eytan R. Barnea, and Nelson Fernandez. "Preimplantation Factor (PIF) Promotes HLA-G, -E, -F, -C Expression in JEG-3 Choriocarcinoma Cells and Endogenous Progesterone Activity." Cellular Physiology and Biochemistry 43, no. 6 (2017): 2277–96. http://dx.doi.org/10.1159/000484378.
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Background/Aims: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF), a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and –C) and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells. Methods: PIF and progesterone (P4) effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. Results: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs), coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1), resulting in improved maternal signalling. Conclusion: PIF can generate a pro-tolerance milieu by enhancing the expression of HLA molecules and by amplifying endogenous progesterone activity. A Fast-Track clinical trial for autoimmune disease has been satisfactorily completed. The acquired data warrants PIF use for the treatment of early pregnancy disorders.
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Na, Brian, C. Dustin Rubinstein, Jennifer J. Meudt, Jaclyn A. Biegel, Alexander R. Judkins, Brent P. Lehman, Jamie L. Reichert, Jeremie Vitte, Dhanansayan Shanmuganayagam, and Marco Giovannini. "MODL-13. GENETICALLY ENGINEERED PIG MODEL OF RHABDOID TUMOR PREDISPOSITION SYNDROME-1." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii413. http://dx.doi.org/10.1093/neuonc/noaa222.587.
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Abstract Atypical teratoid/rhabdoid tumor (AT/RT) is the most common malignant CNS tumor of children below 6 months of age. The majority of AT/RT demonstrate genomic alterations in the SMARCB1 gene. There are two major hurdles in the development of safe and effective treatments for AT/RT: first, the mouse models do not fully recapitulate the disease seen in patients and their predictivity of clinical efficacy is still unproven. Second, due to a small patient population, the ability to recruit enough patients for clinical trials is challenging. Genetic studies have demonstrated that germline deletion of SMARCB1 exons 4 and 5 predisposes to AT/RT at an early age. Comparison of human, swine, and mouse SMARCB1 genes show similarities in gene and protein structure, with 100% amino acid identity between swine and human SMARCB1 isoforms. Thus, we hypothesized that germline deletion of exons 4 and 5 will predispose heterozygote swine to AT/RT development. SMARCB1+/- founder pigs are obtained using a CRISPR/Cas9 mediated gene-editing of conventional crossbred swine embryos, followed by embryo transfer into female swine surrogates. They are evaluated for clinical criteria used to diagnose AT/RT and by MRI at 6, 12, and 24 months of age, followed by histopathology and molecular analysis of the tumors as they are detected. Generating a large animal model of AT/RT would represent a breakthrough in the field from a genomic, pathophysiologic, pre-clinical and therapeutic perspective.
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Han, Shao-Cong, Rong-Ping Huang, Qiong-Yi Zhang, Chang-Yu Yan, Xi-You Li, Yi-Fang Li, Rong-Rong He, and Wei-Xi Li. "Antialcohol and Hepatoprotective Effects of Tamarind Shell Extract on Ethanol-Induced Damage to HepG2 Cells and Animal Models." Foods 12, no. 5 (March 3, 2023): 1078. http://dx.doi.org/10.3390/foods12051078.
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Alcohol liver disease (ALD) is one of the leading outcomes of acute and chronic liver injury. Accumulative evidence has confirmed that oxidative stress is involved in the development of ALD. In this study, we used chick embryos to establish ALD model to study the hepatoprotective effects of tamarind shell exttract (TSE). Chick embryos received 25% ethanol (75 μL) and TSE (250, 500, 750 μg/egg/75 μL) from embryonic development day (EDD) 5.5. Both ethanol and TSE were administrated every two days until EDD15. Ethanol-exposed zebrafish and HepG2 cell model were also employed. The results suggested that TSE effectively reversed the pathological changes, liver dysfunction and ethanol-metabolic enzyme disorder in ethanol-treated chick embryo liver, zebrafish and HepG2 cells. TSE suppressed the excessive reactive oxygen species (ROS) in zebrafish and HepG2 cells, as well as rebuilt the irrupted mitochondrial membrane potential. Meanwhile, the declined antioxidative activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD), together with the content of total glutathione (T-GSH) were recovered by TSE. Moreover, TSE upregulated nuclear factor erythroid 2—related factor 2 (NRF2) and heme oxyense-1 (HO-1) expression in protein and mRNA level. All the phenomena suggested that TSE attenuated ALD through activating NRF2 to repress the oxidative stress induced by ethanol.
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Okuda, Kazuhide S., Mei Fong Ng, Nur Faizah Ruslan, Neil I. Bower, Dedrick Soon Seng Song, Huijun Chen, Sungmin Baek, et al. "3,4-Difluorobenzocurcumin Inhibits Vegfc-Vegfr3-Erk Signalling to Block Developmental Lymphangiogenesis in Zebrafish." Pharmaceuticals 14, no. 7 (June 26, 2021): 614. http://dx.doi.org/10.3390/ph14070614.
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Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.
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Zhou, Qi, Yan Zhang, Yujie Zou, Tailang Yin, and Jing Yang. "Human embryo gene editing: God's scalpel or Pandora's box?" Briefings in Functional Genomics 19, no. 3 (February 26, 2020): 154–63. http://dx.doi.org/10.1093/bfgp/elz025.
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Abstract Gene editing refers to the site-specific modification of the genome, which mainly focuses on basic research, model organism construction and treatment and prevention of disease. Since the first application of CRISPR/Cas9 on the human embryo genome in 2015, the controversy over embryo gene editing (abbreviated as EGE in the following text) has never stopped. At present, the main contradictions focus on (1) ideal application prospects and immature technologies; (2) scientific progress and ethical supervision; and (3) definition of reasonable application scope. In fact, whether the EGE is ‘God's scalpel’ or ‘Pandora's box’ depends on the maturity of the technology and ethical supervision. This non-systematic review included English articles in NCBI, technical documents from the Human Fertilization and Embryology Authority as well as reports in the media, which performed from 1980 to 2018 with the following search terms: ‘gene editing, human embryo, sequence-specific nuclease (SSN) (CRISPR/Cas, TALENT, ZFN), ethical consideration, gene therapy.’ Based on the research status of EGE, this paper summarizes the technical defects and ethical controversies, enumerates the optimization measures and looks forward to the application prospect, aimed at providing some suggestions for the development trend. We should regard the research and development of EGE optimistically, improve and innovate the technology boldly and apply its clinical practice carefully.