Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47.877
Filtrar
3.
Theriogenology ; 173: 93-101, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34365139

RESUMO

Cryopreservation of both gametes and embryos, both for storage and for the preservation of their developmental capacity is a critical aspect of assisted reproductive technology. The survival of reproductive material following cryopreservation protocols is not only vital to clinical applications in the human in vitro fertilisation clinic, but is also important in the in vitro production of livestock embryos. The ability to routinely cryopreserve oocytes and embryos of livestock species has the potential to improve animal welfare, reduce environmental impact, and reduce the associated costs for breeding companies through the reduction of live animal transportation. Unfortunately, frozen oocytes and embryos are regularly documented to contain a higher proportion of apoptotic cells compared to their non-frozen counterparts, with freezing procedures thought to trigger apoptotic pathways of cell death. Comparisons between frozen and non-frozen samples also show changes in the gene expression of apoptotic factors such as Bcl-2 and Bax in response to cryopreservation. Apoptotic inhibition has the potential to improve cryosurvival, and how to achieve this is subject to debate. Here, we review how exposure to low temperatures during cryopreservation may be responsible for the abnormal activation of apoptotic pathways in mammalian oocytes and embryos, and discuss the ways in which they can be influenced to improve cryopreservation protocols, particularly in agriculturally important species.


Assuntos
Criopreservação , Embrião de Mamíferos , Animais , Apoptose , Criopreservação/veterinária , Fertilização In Vitro/veterinária , Oócitos
4.
Theriogenology ; 173: 123-127, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34371439

RESUMO

A total of 184 dromedary camel embryos were vitrified using a novel vitrification kit specifically developed for camel embryos. These embryos were vitrified using a 3-step process by exposing them to vitrification solutions (VS) containing 20% foetal calf serum (FCS) with (+) or without (-) the addition of bovine serum albumin (BSA). Embryos were then further divided into two groups (

Assuntos
Camelus , Vitrificação , Animais , Criopreservação/veterinária , Embrião de Mamíferos , Feminino , Gravidez , Taxa de Gravidez
5.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445470

RESUMO

In regular IVF, a portion of oocytes exhibit abnormal numbers of pronuclei (PN) that is considered as abnormal fertilization, and they are routinely discarded. However, it is known that abnormal ploidy still does not completely abandon embryo development and implantation. To explore the potential of cytoplasm from those abnormally fertilized oocytes, we developed a novel technique for the transfer of large cytoplasm between pronuclear-stage mouse embryos, and assessed its impact. A large volume of cytoplast could be efficiently transferred in the PN stage using a novel two-step method of pronuclear-stage cytoplasmic transfer (PNCT). PNCT revealed the difference in the cytoplasmic function among abnormally fertilized embryos where the cytoplasm of 3PN was developmentally more competent than 1PN, and the supplementing of fresh 3PN cytoplasm restored the impaired developmental potential of postovulatory "aged" oocytes. PNCT-derived embryos harbored significantly higher mitochondrial DNA copies, ATP content, oxygen consumption rate, and total cells. The difference in cytoplasmic function between 3PN and 1PN mouse oocytes probably attributed to the proper activation via sperm and may impact subsequent epigenetic events. These results imply that PNCT may serve as a potential alternative treatment to whole egg donation for patients with age-related recurrent IVF failure.


Assuntos
Núcleo Celular/patologia , Citoplasma/patologia , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Zigoto/patologia , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICR , Zigoto/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445775

RESUMO

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.


Assuntos
Ciclo Celular/genética , Cromatina/genética , Segregação de Cromossomos/genética , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Inativação Gênica/fisiologia , Transcrição Genética/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos
7.
Theriogenology ; 174: 131-138, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34450564

RESUMO

The equine embryo or embryonic vesicle on Days 11-15 postovulation travels with profound physiologic purpose throughout the lumen of the two uterine horns and uterine body making 12 to 22 trips between the two uterine horns per day. This phenomenon is termed embryo mobility and is unique in equids among domestic species. Apparently, the embryo first reaches the uterine body on Days 8 or 9. Mobility increases to maximum by Days 11 or 12 and continues until an abrupt cessation of mobility (fixation) on Days 15 (ponies) or 16 (horses and donkeys). The embryo is propelled by uterine contractions in response to the production of apparently both PGF2α and PGE2 by both the embryo and uterus. An increase in endometrial vascular perfusion accompanies the mobile embryo as it moves from horn to horn. Restricting the embryo to one uterine horn by a ligature has indicated that specific roles of the traveling embryo include the stimulation of uterine contractions, tone, vascularity, and edema and to curtail the production of the luteolysin (PGF2α) by the uterus. The increase in uterine tone, decrease in diameter of the uterine horns, and a flexure in the caudal portion of each horn collaborate in the selection of a horn of fixation. Embryo mobility is a game changer that has solved several long-time enigmas in mare reproduction and has provided a needed and effective finger/thumb compression method for eliminating one member of a twin set.


Assuntos
Embrião de Mamíferos , Endométrio , Animais , Equidae , Feminino , Cavalos , Gravidez , Contração Uterina , Útero
8.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
9.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361119

RESUMO

Developmental arrest of the preimplantation embryo is a multifactorial condition, characterized by lack of cellular division for at least 24 hours, hindering the in vitro fertilization cycle outcome. This systematic review aims to present the molecular drivers of developmental arrest, focusing on embryonic and parental factors. A systematic search in PubMed/Medline, Embase and Cochrane-Central-Database was performed in January 2021. A total of 76 studies were included. The identified embryonic factors associated with arrest included gene variations, mitochondrial DNA copy number, methylation patterns, chromosomal abnormalities, metabolic profile and morphological features. Parental factors included, gene variation, protein expression levels and infertility etiology. A valuable conclusion emerging through critical analysis indicated that genetic origins of developmental arrest analyzed from the perspective of parental infertility etiology and the embryo itself, share common ground. This is a unique and long-overdue contribution to literature that for the first time presents an all-inclusive methodological report on the molecular drivers leading to preimplantation embryos' arrested development. The variety and heterogeneity of developmental arrest drivers, along with their inevitable intertwining relationships does not allow for prioritization on the factors playing a more definitive role in arrested development. This systematic review provides the basis for further research in the field.


Assuntos
Blastocisto/patologia , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Fertilização In Vitro , Humanos
10.
J Int Bioethique Ethique Sci ; Vol. 32(1): 95-111, 2021 May 24.
Artigo em Francês | MEDLINE | ID: mdl-34378886

RESUMO

In Belgian law, the granting of legal personality presupposes the meeting of three conditions being born, alive and viable.The embryo therefore does not have this personality, namely the ability to acquire rights and obligations.Any other solution would have unacceptable consequences, whether for the right of women to voluntary termination of the pregnancy or for the future of research and medical acts involving the use of the embryo: medically assisted procreation, embryo donation, embryo research …However, the embryo cannot be totally assimilated to a thing in view of its biological existence.This difference between legal personality and human personality leads the legislator to surround all manipulations of the embryo with guarantees and conditions intended to ensure a respectful framework for this potentiality of life.


Assuntos
Destinação do Embrião , Pesquisas com Embriões , Bélgica , Embrião de Mamíferos , Feminino , Humanos , Gravidez
11.
Nat Commun ; 12(1): 4856, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381034

RESUMO

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Reprogramação Celular , Células-Tronco Totipotentes/citologia , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Morte Celular , Dano ao DNA , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Células-Tronco Totipotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 12(1): 4171, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234116

RESUMO

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Saxitoxina/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células CHO , Células Cultivadas , Corpo Caloso/citologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Cricetulus , Embrião de Mamíferos , Feminino , Hipocampo/citologia , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/efeitos da radiação , Análise de Célula Única , Análise Espaço-Temporal , Raios Ultravioleta , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos da radiação
13.
Nat Commun ; 12(1): 4208, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244516

RESUMO

The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFß and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFß-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.


Assuntos
Fibrocartilagem/crescimento & desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Tendões/crescimento & desenvolvimento , Engenharia Tecidual/métodos , Ativação Transcricional , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Fibrocartilagem/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Mecanotransdução Celular/genética , Camundongos , RNA-Seq , Transdução de Sinais/genética , Análise de Célula Única , Tendões/citologia , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/metabolismo
14.
J R Soc Interface ; 18(180): 20210109, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34283940

RESUMO

During development, progenitor cells follow timetables for differentiation that span many cell generations. These developmental timetables are robustly encoded by the embryo, yet scalably adjustable by evolution, facilitating variation in organism size and form. Epigenetic switches, involving rate-limiting activation steps at regulatory gene loci, control gene activation timing in diverse contexts, and could profoundly impact the dynamics of gene regulatory networks controlling developmental lineage specification. Here, we develop a mathematical framework to model regulatory networks with genes controlled by epigenetic switches. Using this framework, we show that such epigenetic switching networks uphold developmental timetables that robustly span many cell generations, and enable the generation of differentiated cells in precisely defined numbers and fractions. Changes to epigenetic switching networks can readily alter the timing of developmental events within a timetable, or alter the overall speed at which timetables unfold, enabling scalable control over differentiated population sizes. With their robust, yet flexibly adjustable nature, epigenetic switching networks could represent central targets on which evolution acts to manufacture diversity in organism size and form.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Diferenciação Celular , Embrião de Mamíferos , Epigênese Genética
15.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34269385

RESUMO

Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.


Assuntos
Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Endossomos/metabolismo , Feminino , Fertilização , Proteínas da Membrana Plasmática de Transporte de Glicina , Masculino , Camundongos , Oócitos , Proteína Quinase C , Ubiquitina/metabolismo , Ubiquitinação
16.
Theriogenology ; 173: 37-47, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34329894

RESUMO

Reproducing the environment to which the embryo is naturally exposed may be an alternative to improve viability of embryos produced in vitro. In the first part of this work, we describe a novel culture media, namely Embryonic Culture Supplementation (ECS100). The composition of this media was based on the contents of carbohydrates and amino acids found in oviductal and uterine fluids. Because it was a new formulation, we investigated the performance of ECS100 in comparison with conventionally used SOFaa, and possible benefits to embryo development. Embryo production rates (cleavage, morula and blastocyst conversion, blastocyst and hatching rates) and morphophysiological parameters (total cell number, cell allocation, Mitochondrial membrane potential (MMP), Reactive Oxygen Species (ROS), NADH, FAD+ and ATP content) were similar between ECS100 and SOFaa. Next, we tested if a reduction of ECS100 concentration could positively contribute to embryo viability by resembling the more dynamic availability of nutrients that reach the embryos in vivo. Therefore, embryos were cultured in ECS100 or in its serial dilution (ECS75, 50 and 25). Despite the fact that the lowest concentration (ECS25) still supported blastocyst formation, halving the concentration of metabolites (ECS50) actually improved embryo production rates. Thus, embryos produced in ECS100 or ECS50 were submitted to further analyses on Days 4 and 7. Embryos cultured in ECS50 presented better developmental rates and morphophysiological profile than embryos cultured in ECS100. Additionally, physiological traits (MMP, ROS and NADH levels) of embryos cultured in ECS50 presented the expected pattern for embryos produced in vivo. In conclusion, we presented a novel, more personalized and effective culture media for bovine IVP embryos. And although the ECS media formulation was based on the contents of female reproductive fluids, it is worth mentioning that adaptations must be specifically directed for in vitro conditions rather than reproduced exactly from in vivo state.


Assuntos
Blastocisto , Técnicas de Cultura Embrionária , Animais , Bovinos , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Fertilização In Vitro/veterinária , Nutrientes
17.
Cell Prolif ; 54(8): e13097, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34250657

RESUMO

OBJECTIVES: Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. MATERIALS AND METHODS: To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. RESULTS: Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. CONCLUSIONS: Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.


Assuntos
Desenvolvimento Embrionário , Fatores de Transcrição SOXB1/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , RNA Guia/metabolismo , Fatores de Transcrição SOXB1/genética , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
18.
Methods Mol Biol ; 2319: 93-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331247

RESUMO

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.


Assuntos
Angiografia/métodos , Vasos Sanguíneos/diagnóstico por imagem , Técnicas de Cultura/métodos , Embrião de Mamíferos/diagnóstico por imagem , Desenvolvimento Embrionário , Microscopia de Fluorescência/métodos , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Meios de Cultura/química , Dissecação/métodos , Embrião de Mamíferos/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Microscopia Confocal
19.
Methods Mol Biol ; 2319: 119-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331250

RESUMO

We describe a novel, efficient method to identify cis-acting DNA sequences that drive cell-specific gene expression during development. We utilize transfer of Bacterial Artificial Chromosome (BAC) genomic DNAs, modified to contain a reporter gene, into fertilized mouse embryos and placing the injected embryos into pseudopregnant recipient females. The embryos are allowed to develop in utero for defined times after which they are collected for analysis. Using DNAs containing the LacZ reporter gene facilitates the analysis of gene activity through microscopy of intact embryos and subsequent sectioning of the stained embryos. With this technique cis-element activity can be identified and evaluated through further mutational analysis of the injected BAC DNA. This allows the identification of important gene regulatory domains that specify stage-specific gene expression in the developing embryo.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Embrião de Mamíferos/diagnóstico por imagem , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Embrião de Mamíferos/metabolismo , Feminino , Genes Reporter/genética , Camundongos , Camundongos Transgênicos , Microinjeções/métodos , Recombinação Genética , beta-Galactosidase/genética
20.
Methods Mol Biol ; 2319: 153-159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331253

RESUMO

Pathological alterations of lymphatic structure and function interfere with lymph transport, resulting in a wide range of clinical disorders that include edema, tissue inflammation, and metabolic syndromes. Mesentery contains abundant lymphatic vessels and plays an important role in transporting absorbed lipid from the intestine. In this manuscript, we describe a whole-mount staining method on isolated mouse mesentery with VEGFR3, Prox1, and Lyve1 antibodies to visualize the morphology of lymphatic vessels.


Assuntos
Linfangiogênese , Vasos Linfáticos/metabolismo , Mesentério/citologia , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Animais , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Mesentério/metabolismo , Camundongos , Proteínas Supressoras de Tumor/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...