Name: the chick chorioallantoic membrane (cam) model as a tool to study ovarian tissue transplantation
Uploaded: 2023-06-23T14:30:00
Description: Watch this Scientific Journal Video about The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation at JoVE.com

The authors thank Mira Hryniuk, BA, for reviewing the English language of the article.

The use of human tissue was approved by the Institutional Review Board of the Catholic University of Louvain. The patients gave their written informed consent for the use of their ovarian tissue for research purposes.
1. Ordering day 0 eggs that are highly likely to be embryonated
<ol>
	<li>Find a certified laboratory-grade Lohman-selected white Leghorn egg supplier that reports high rates of embryonated eggs, which is primarily dependent on the age of the chicks.</li>
</ol>...

The most challenging part of the protocol described here is making the small hole required to aspirate the albumen in order to detach the CAM from the eggshell prior to creating a window. Applying too much pressure can result in overpenetration or may even crack and destroy the egg, causing irrevocable damage to the CAM and its vasculature. To keep mistakes to a minimum during initial attempts to separate the CAM, it is strongly advised to practice making small holes in the eggshell of non-fertilized, grocery-bought eggs...

The demand for fertility preservation for oncological and benign indications, as well as social reasons, has dramatically increased over recent decades. However, various treatments used to cure malignant and non-malignant diseases are highly toxic to the gonads and can result in iatrogenic premature ovarian insufficiency, ultimately leading to infertility1. Established techniques for fertility preservation include embryo cryopreservation, immature or mature oocyte vitrification, and ovarian tissue cryopreservation2,3,4. Ovarian tissue freezing is the only available option for preserving fertility in prepubertal girls or women who require immediate cancer therapy. The restoration of endocrine function following ovarian tissue transplantation occurs in over 95% of subjects, with live birth rates ranging from 18% to 42%5,6,7,8,9.
Although the transplantation of frozen-thawed ovarian tissue has proven successful, there is still room for improvement. Indeed, as ovarian cortical fragments are transplanted without vascular anastomosis, they experience a period of hypoxia during which graft revascularization takes place10,11,12. The vast majority of studies investigating human ovarian tissue transplantation have used a xenografting model, in which ovarian tissue is transplanted to immunodeficient mice. The complete revascularization of the xenografts takes around 10 days, with both the host and graft vessels contributing to the formation of functional vessels12,13,14. Around 50%-90% of the follicle reserve is lost during this hypoxic window before the completion of graft revascularization10,15,16. It has been strongly suggested that this massive follicle loss occurs through both direct follicle death, as demonstrated by a decrease in the absolute follicle numbers left after grafting, and the activation of primordial follicle growth, as indicated by changes in follicle proportions towards increased rates of growing follicles17,18.
Interestingly, previous research works using various animal ovarian tissues grafted to chick chorioallantoic membrane (CAM), which has a constitution mimicking the typical grafting site of the peritoneum, have reported the inhibition of spontaneous follicle activation, with the primordial follicle reserve staying intact for up to 10 days19,20,21,22. Our team&#160;previously demonstrated that the grafting of frozen-thawed human ovarian tissue to CAM constituted a reliable approach for investigating human ovarian tissue transplantation in its first ischemic stages23&#160;and recently showed that this grafting method was able to counteract follicle activation24.
The CAM model is especially appealing not only because eggs are much cheaper than mice but also because of the highly vascularized nature of CAM, allowing scrutiny of the association between follicle activation and ovarian graft revascularization. The avian system is, indeed, one of the most common and versatile ways of studying angiogenesis25. Chick embryo development (ED) takes 21 days until hatching, and the CAM is formed within the first 4-5 days through the fusion of the allantois and chorion26. Notably, the chick embryo is a naturally immunodeficient host until day 17 of ED, so xenografting experiments can be performed without any risk of graft rejection27,28. Moreover, the CAM model approach does not raise any ethical or legal concerns in terms of European law29, making it an attractive alternative to other animal models. With regard to breeding conditions, chick embryos only need an incubator set at 37 &#176;C with a relative air humidity of 40%-60%. These limited experimentation requirements significantly reduce the research costs compared to use of immunodeficient mice.
The protocol presented herein aims to describe the development of a CAM xenografting model for human ovarian tissue and provide specific insights into the effectiveness of the technique, the time frame of graft revascularization, and the tissue viability over a 6 day grafting period. This protocol could be of great interest for investigating the mechanisms behind early post-grafting follicle loss and studying the impact of several agents (growth factors, hormones, etc.) on this phenomenon.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agani hypodermic needle, 19 G</td>
			<td>Terumo Europe</td>
			<td>AN*1950R1</td>
			<td>19 G needle to aspirate albumen</td>
		</tr>
		<tr>
			<td>Terumo syringe, 5 mL concentric Luer lock</td>
			<td>Terumo Europe</td>
			<td>SS*05LE1</td>
			<td>5-mL sterile syringe</td>
		</tr>
		<tr>
			<td>Caseviewer v2.2</td>
			<td>3DHISTECH</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Merck Chemicals</td>
			<td>603-022-00-4</td>
			<td>Sterile ether to traumatize the CAM</td>
		</tr>
		<tr>
			<td>Eosin Y aqueous solution 0.5%</td>
			<td>Merck</td>
			<td>1098441000</td>
			<td>Staining solution</td>
		</tr>
		<tr>
			<td>Formaldehyde 4% aqueous solution buffered (Formalin 10%)</td>
			<td>VWR</td>
			<td>97139010</td>
			<td>Formaldehyde used for tissue fixation</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Liebherr</td>
			<td>7081260</td>
			<td>Fridge at 4 &#176;C used for paraffin-embedding</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Schott</td>
			<td>SLK2</td>
			<td>Hot plate used to dry the slides</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Forma Scientific 3111</td>
			<td>10365156</td>
			<td>Oven used for slide incubation</td>
		</tr>
		<tr>
			<td>Leica CLS 150 XE microscope cold light source</td>
			<td>Leica</td>
			<td>CLS 150 XE</td>
			<td>Focal cold light source to candle the eggs</td>
		</tr>
		<tr>
			<td>Lens cleaning tissue, grade 541</td>
			<td>VWR</td>
			<td>111-5003</td>
			<td>Tissue to soak in sterile ether to traumatize the CAM</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hematoxylin</td>
			<td>MERCK</td>
			<td>1092491000</td>
			<td>Staining solution</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20847307</td>
			<td>Methanol</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>ThermoScientific-MICROM</td>
			<td>HM325-2</td>
			<td>Microtome</td>
		</tr>
		<tr>
			<td>Pannoramic P250 Flash III</td>
			<td>3DHISTECH</td>
			<td>/</td>
			<td>Slide scanner at 20x magnification</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1,04,00,51,000</td>
			<td>Paraffin-embedding solution</td>
		</tr>
		<tr>
			<td>Paraplast Plus R</td>
			<td>Sigma</td>
			<td>P3683-1KG</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>Petri dish, 60x15 mm, sterile</td>
			<td>Greiner</td>
			<td>628161</td>
			<td>Sterile petri dish</td>
		</tr>
		<tr>
			<td>Pin holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>Pin holder</td>
		</tr>
		<tr>
			<td>Polyhatch</td>
			<td>Brinsea</td>
			<td>CP01F</td>
			<td>Egg incubator with automatic rotator</td>
		</tr>
		<tr>
			<td>Scroll saw blade, 132 mm</td>
			<td>Sencys</td>
			<td>/</td>
			<td>Saw blade to create a window in the eggshell</td>
		</tr>
		<tr>
			<td>Stainless steel insert pins</td>
			<td>Fine Science Tools</td>
			<td>26007-02</td>
			<td>Straight pin to make a hole in the eggshell</td>
		</tr>
		<tr>
			<td>Steril-Helios&#160;</td>
			<td>Angelantoni Industrie</td>
			<td>ST-00275400000</td>
			<td>Laminar flow hood</td>
		</tr>
		<tr>
			<td>Superfrost Plus bords rod&#233;s 90&#176;</td>
			<td>VWR</td>
			<td>631-9483</td>
			<td>Glass slides&#160;</td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 6Al</td>
			<td>Sakura</td>
			<td>60320417-0711 VID6E3-1</td>
			<td>Automatic embedding device</td>
		</tr>
		<tr>
			<td>Titanium forceps</td>
			<td>Fine Science Tools</td>
			<td>11602-16</td>
			<td>Forceps for eggshell removal and ovarian tissue manipulation</td>
		</tr>
		<tr>
			<td>Toluene, pa</td>
			<td>VWR</td>
			<td>28701364</td>
			<td>Paraffin-embedding solution</td>
		</tr>
	</tbody>
</table>

the chick chorioallantoic membrane (cam) model as a tool to study ovarian tissue transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Chick embryo survival rates 
The embryo survival rate from windowing (day 3 of&#160;ED) to ovarian tissue grafting (day 7 of ED) was 79% (33/42). Since the percentage of embryonated day 0 eggs is unknown, supernumerary day 0 eggs from Lohman-selected white Leghorn chickens were ordered to ensure sufficient embryonated eggs would be available for grafting. A total of 23 viable day 7 eggs were used for grafting, one of which perished during the first 24 h, resulting in an overall embryo survival rate ...

Watch this Scientific Journal Video about The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation at JoVE.com

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

The use of CAM to observe early changes after ovarian tissue transplantation has a significant advantage over in vitro models, since it allows the investigation of mechanism affecting the early vascularization and follicle loss process. This technique is inexpensive, and can be performed without any risk of graft rejection due to the natural embryo immunodeficiency for the first 17 days. Moreover, this approach does not raise any ethical or legal concerns in terms of European law.The best strategy to develop the manual skills needed for egg manipulation may be reached by training with non-embryonated eggs before the actual experiment. To begin, label the day zero eggs received from Louvain selected white leghorn chicks using a marker. Incubate the eggs in an incubator set at 37 degrees celsius, with the pointed ends facing down.Rotate the eggs by 180 degrees, two to three times daily manually. In the dark, place an egg candler against the eggshell and pinpoint the center of the air pocket on the eggshell using a marker. Turn on the lights and rotate a sterile straight pin at the marked position to make an opening of around one millimeter.Locate the yolk sack in the dark using the egg candler. Puncture the egg with a sterile 19 gauge needle, angled at 45 degrees toward the bottom of the egg, without disrupting the yolk sack. Aspirate 1.5 to two milliliters of the albumin to detach the CAM from the shell.Then close the hole with adhesive tape. Turn on the lights and draw a rectangular window measuring one by 1.5 centimeters on the horizontally placed egg. Hold the egg in one hand, and gently cut a window into the eggshell, using a scroll saw blade, without cracking the shell.Blow regularly to remove shell dust and debris. Slide sterile forceps under the rectangular piece of shell, and remove it cleanly without damaging the CAM. Then discard the white outer shell membrane to see the embryo and the CAM.In viable embryonated eggs, clear albumin, a vascular ring around the embryo, and sometimes a beating heart, are seen on day three of chick embryo development. Under the laminar flow hood, place the egg on the egg rack with the window facing up and peel off the tape. Gently place and immediately remove a one centimeter square strip of sterile ether-extracted lens paper onto the epithelial surface, to traumatize a small area of the CAM.Then grasp one frozen thawed ovarian cortical strip with microsurgical forceps, and place it onto the traumatized CAM with the medullary side against the CAM. Evaluate the graft macroscopically, and pay particular attention to the vascular reaction of the CAM toward the graft before capturing digital photographs or videos. Once done, harvest the graft by grasping the tissue or the surrounding CAM with forceps, and carefully excising the graft from the CAM using scissors.The grafts were viable in 100%of cases after the first day of the grafting. They were already partially adherent to the CAM, and showed the wheel spoke pattern of blood vessels needed for their vascularization. After six days of grafting, the implants were adhered to the CAM.Around day three post-transplantation, the grafts were covered with a second layer of CAM and became encapsulated. The graft showed good vascularization, with 80%of the transplants penetrating the egg. Two grafts did not attach to the CAM and took on a necrotic appearance, resulting in a grafted tissue survival rate of 83%after six days.In the histological evaluation of the transplant, healthy follicles were observed at all time points. After the transplantation, an insignificant decrease was observed in the follicle densities. The follicle survival rates were maintained during the grafting period at 95%on day one, and 83%on day six.The most challenging part is making the small hole required to aspirate the albumin, to detach the CAM from the eggshell, before creating a window applying. Too much pressure can result in overstimulation, or may even crack and destroy the egg, causing irrevocable damage to the CAM or its vasculature. This model is ideal for testing several aspects of the early ovarian tissue post-grafting period, including the impact of pro-angiogenic factors, protective antioxidant agents, cytokines, hormones, and other factor controlling follicle activation and early growth.This model indicated the link between early vascularization and modulation of follicle activation, which are the two main events resulting in the ovarian reserve decline after ovarian tissue transplantation.

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Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...