A protocol for in ovo electroporation of chicken and snake embryos to study forebrain development.
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ABSTRACT:
SUBMITTER: Cardenas A
PROVIDER: S-EPMC8339381 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 ?m thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.
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Project description:Cancer metastasis is the leading cause of cancer-related mortality worldwide. To date, several in vitro methodologies have been developed to understand the mechanisms of cancer metastasis and to screen various therapeutic agents against it. Nevertheless, mimicking an in vivo microenvironment in vitro is not possible; while in vivo experiments are complex, expensive and bound with several regulatory requirements. Herein, we report a novel in ovo model that relies on chicken embryo to investigate cancer cell invasion and metastasis to various organs of the body. In this model, we directly injected green fluorescent protein (GFP) expressing cancer cells to the heart of chicken embryo at 3 days of incubation, then monitored cell migration to various organs. To this end, we used a simple tissue processing technique to achieve rapid imaging and quantification of invasive cells. We were able to clearly observe the migration of GFP expressing cancer cells into various organs of chicken embryo. Organ specific variation in cell migration was also observed. Our new slide pressing based tissue processing technique improved the detectability of migrated cells. We herein demonstrate that the use of GFP expressing cancer cells allows easy detection and quantification of migrated cancer cells in the chicken embryo model, which minimizes the time and effort required in this types of studies compared to conventional histopathological analysis. In conclusion, our investigation provides a new cancer metastasis model that can be further improved to include more complex aspects, such as the use of multiple cell lines and anti-metastatic agents, thus opening new horizons in cancer biology and pharmaceutical research.
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Project description:BackgroundThe chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging.MethodsIn the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters.ResultsTransgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4.ConclusionWe demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.
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Project description:We have examined the effects of the synthetic cannabinoids HU 210 and HU 211, the plant-derived cannabidiol and the endogenous cannabinoid anandamide on the viability and development of chick embryos. Fertilized White Leghorn chicken eggs were injected with the test compounds or carrier vehicle, via a drilled small hole in the egg, directly into the egg yolk. After nine days of exposure, the embryonal viability, length and wet weight of embryos, and wet weight of brains were measured, and the development stages were assessed according to the Hamburger and Hamilton (HH) scale. The potent synthetic cannabinoid receptor agonist HU 210 and the non-psychotropic cannabidiol were embryotoxic at the highest concentrations examined (10 µM and 50 µM, respectively), with no viable embryos after the HU 210 injection, and 20% viability after the cannabidiol injections. The effects of HU 210 on the chick embryo were attenuated by α-tocopherol and the cannabinoid receptor antagonist AM251, whereas only α-tocopherol gave a statistically significant protection against the embryotoxic effects of cannabidiol. This study shows that exposure to plant-derived or synthetic cannabinoids during early embryonal development decreases embryonal viability. Extrapolation of data across species is of course difficult, but the data would argue against the use of cannabinoids, be it recreationally or therapeutically, during pregnancy.
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Project description:Fertilized chicken eggs were injected with environmental doses of 4 chiral polychlorinated biphenyls (PCBs) and 8 polybrominated biphenyl ethers (PBDEs) to investigate their uptake, metabolism in the embryo, and distribution in the neonate chicken. PCB95 uptake was the most efficient (80%) whereas BDE209 was the least (56%). Embryos metabolized approximately 52% of the PCBs absorbed. Though some degree of metabolism in the first 18 days, most of the PCBs and PBDEs was metabolized in the last three days, when BDE85, 99, 153, and 209 decrease by 11-37%. Enantioselective metabolism of the (+) enantiomers of PCB95, 149, and 132 and the (-) enantiomer of PCB91 was observed. The enantioselective reactivity was higher with the two penta-PCBs than the two tetra-PCBs. Liver, exhibited high affinity for high lipophilic chemicals, enrich all chemicals that was deflected in other tissues except for some special chemicals in a given tissues. Lipid composition, time of organ formation, and metabolism contribute to the distribution of chemicals in the neonate chicken. The result of this study will improve our understanding on the fate and potential adverse effects of PCBs and PBDEs in the neonate chicken.
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Project description:While the correlation between diabetes during pregnancy and birth defects is well-established, how hyperglycemia causes developmental abnormalities remains unclear. In this study, we developed a novel "hyperglycemic" chicken embryonic model by administrating various doses of glucose to fertilized eggs at embryonic stages HH16 or HH24. When the embryos were collected at HH35, the LD50 was 1.57 g/Kg under HH16 treatment and 0.93 g/Kg under HH24 treatment, indicating that "hyperglycemic" environments can be lethal for the embryos. When exposed to a dose equal to or higher than 1 g/Kg glucose at HH16 or HH24, more than 40% of the surviving chicken embryos displayed heart defects and/or limb defects. The limb defects were associated with proliferation defects of both the wing and leg buds indicated by reduced numbers of p-H3S10 labeled cells. These limb defects were also associated with ectopic apoptosis in the leg bud and expression changes of key apoptotic genes. Furthermore, glucose treatment induced decreased expression of genes involved in Shh-signaling, chondrogenesis, and digit patterning in the limb bud. In summary, our data demonstrated that a high-glucose environment induces congenital heart and limb defects associated with disrupted cell proliferation and apoptosis, possibly through depressed Shh-signaling.
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Project description:BackgroundRetinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete.ResultsThis study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types.ConclusionThe new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.
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Project description:The culling of day-old male chicks has caused ethical and economic concerns. Traditional approaches for detecting the in ovo sex of chicken embryos involve opening the eggshell and inner membrane, which are destructive, time-consuming, and inefficient. Therefore, noncontact optical sensing techniques have been examined for the in ovo sexing of chicken embryos. Compared with traditional methods, optical sensing can increase determination throughput and frequency for the rapid sexing of chicken embryos. This paper presented a comprehensive review of the different optical sensing techniques used for the in ovo sexing of chicken embryos, including visible and near-infrared (Vis-NIR) spectroscopy, hyperspectral imaging, Raman spectroscopy, fluorescence spectroscopy, and machine vision, discussing their advantages and disadvantages. In addition, the latest research regarding different detection algorithms and models for the in ovo sexing of chicken embryos was summarized. Therefore, this paper provides updated information regarding the optical sensing techniques that can be used in the poultry industry and related research.
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Project description:Due to their physicochemical and biological properties, silver nanoparticles (NanoAg) have a wide range of applications. In the present study, their roles as a carrier of nutrients and an immunomodulator were tested in chicken embryos. Cysteine (Cys)+NanoAg injected embryos had smaller livers but heavier breasts on the 19th day of embryogenesis. Cys injected embryos had lower oxygen consumption compared to threonine (Thr) or NanoAg injected embryos. The energy expenditure in Thr+NanoAg, or NanoAg injected embryos was higher than Cys or Cys+NanoAg but was not different from uninjected control embryos. Relative expression of the hepatic insulin-like growth factor-I (IGF-I) gene was higher in Cys or NanoAg injected embryos after lipopolysaccharide (LPS) induction. The gene expression of hepatic tumour necrosis factor-alpha (TNF-?) and interleukin-6 (IL-6) did not differ among amino acids, NanoAg and uninjected controls in the non-LPS groups, but increased by many folds in the LPS treated NanoAg, Cys and Cys+NanoAg groups. In LPS treated spleens, TNF-? expression was also up-regulated by NanoAg, amino acids and their combinations, but interleukin-10 (IL-10) expression was down-regulated in Thr, Cys or Thr+NanoAg injected embryos. Toll like receptor-2 (TLR2) expression did not differ in NanoAg or amino acids injected embryos; however, toll like receptor-4 (TLR4) expression was higher in all treated embryos, except for Cys+NanoAg, than in uninjected control embryos. We concluded that NanoAg either alone or in combination with amino acids did not affect embryonic growth but improved immunocompetence, indicating that NanoAg and amino acid complexes can act as potential agents for the enhancement of innate and adaptive immunity in chicken.
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Project description:The major advantage of chicken embryos model is their accessibility for microsurgical manipulations and the dissection of tissues for ex vivo explant culture. Branchial arches are embryonic structure located next to the top of developing heart. Each arch is made of surface ectoderm, endoderm, myogenic mesoderm cells and cranial neural crest cells. The myogenic mesoderm originates from cranial paraxial mesoderm (CPM), which is transiently migrated to branchial arches (BAs). The first branchial arch (BA1) mesoderm contributes to formation of mastication muscles. The second branchial arch (BA2) mesoderm gives rise to facial expression muscles. This article focuses on cell injection in the CPM and bead implantation (gain of function approaches) in the BA2. In order to follow the migration of mesoderm progenitor cells from CPM to BA2, we injected quail cells in the CPM of stage HH10-11 embryos, followed by implantation of SDF-1 bead at stage HH15-16. Later the attraction of quail cells (CXCR4+) towards the SDF-1 source has been observed, using whole-mount immunostaining of a specific quail antibody (QCPN) at stage HH19-22. •Our method, which involves bead implantation followed by quail cell injection, provides useful tools for tracing migratory mesodermal cells in vivo.•The proposed method does not require any commercial kits and can be used for various developmental process.•It does not employ any complicated methods such as genetically engineered permanent cell labeling, multiplicity of fluorescent markers or clonal analysis.
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