eoe sub articles

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissection of Prenatal Embryos
<ol>
	<li>Clean dissecting scissors and forceps with 70% ethanol.</li>
	<li>To collect perinatal embryos at E18.5 or E19.5, euthanize pregnant mice first using CO2&#160;inhalation and then perform cervical dislocation according to ethical guidelines. 
	NOTE: The pregnant female mouse usually gives birth starting at E18.5, however, larger kidneys are easier to manipulate. Therefore, kidneys at E19.5 closer to birth are easier to perform this analysis on. However, pups with bilateral urinary tract obstructions die after birth. Depending on the characteristics of experimental mice, an appropriate collection day/time should be empirically determined.</li>
	<li>Spray 70% ethanol on the ventral abdominal surface and then open the abdominal cavity ventrally using dissecting scissors and forceps.</li>
	<li>Lift the entire uterus and separate it from the body by cutting with dissecting scissors at the tips of the uterine horns.</li>
	<li>Rinse the entire uterus with 1x PBS in a petri dish.</li>
	<li>Cut the uterus segmentally with dissecting scissors and remove placental decidua with dissecting forceps to expose the embryos in yolk sacs.</li>
	<li>Remove the yolk sac first and then remove the amniotic membrane with dissecting forceps to liberate the perinatal embryos.</li>
</ol>

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prenatal mouse embryos retrieval: a procedure to harvest embryos from pregnant mouse

Source: Yun, K. <a href="https://www.jove.com/video/56247" target="_blank">Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection</a>. J. Vis. Exp.&#160;(2017)
This video demonstrates the retrieval of embryos from a pregnant mouse. The recovered perinatal embryos can be used as model systems for studying mammalian development.

Prenatal Mouse Embryos Retrieval: A Procedure to Harvest Embryos from Pregnant Mouse

Source: Yun, K. Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection. J. Vis. Exp.&#160;(2017)
This video demonstrates the ...

Mouse embryos are used as model systems for studying early mammalian embryogenesis. To retrieve perinatal mouse embryos at the desired stage of embryonic development,&#160; begin with a euthanized pregnant mouse in the supine position.
Next, sterilize its abdominal area and make a transverse incision on the abdominal wall to reveal the underlying uterine horns. The uterine horns are long tubular structures containing a string of embryos. Now, carefully separate the uterine horns from the abdomen by excising them from the surrounding tissue.
Transfer the harvested uterine horns into a dish containing buffer. Rinse the uterine horns to remove tissue debris and blood. Further, incise between the implantation sites along the uterine horns to separate each embryo. Excise the decidua, the maternal placental layer, along with the embryo-derived placental disc.
Next, remove the small extraembryonic membranous structure surrounding the embryo that provides nutrition to it - the yolk sac. Finally, remove the amniotic membrane - the innermost extraembryonic membrane consisting of a fluid-filled sac that surrounds and protects the embryo - to release the mouse embryo.
The recovered perinatal embryos can be used for studying early mammalian development.

prenatal-mouse-embryos-retrieval-procedure-to-harvest-embryos-from

Research

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Embryo

2.7K Views. Source: Yun, K. Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection. J. Vis. Exp. (2017) This video demonstrates the retrieval of embryos from a pregnant mouse. The recovered perinatal embryos can be used as model systems for studying mammalian development. 

Video: Prenatal Mouse Embryos Retrieval: A Procedure to Harvest Embryos from Pregnant Mouse - Concept

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

JoVE Journal

Developmental Biology

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Neuroscience

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Prenatal Mouse Embryos Retrieval: A Procedure to Harvest Embryos from Pregnant Mouse

Transcript

Prenatal Mouse Embryos Retrieval: A Procedure to Harvest Embryos from Pregnant Mouse

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme