This work was supported by grants from Research to Prevent Blindness (Career Development Awards to MEZ and ASV and an unrestricted grant to the Department of Ophthalmology), the E. Matilda Zeigler Foundation (MEZ and ASV) and the National Eye Institute/NIH (MEZ, grants R01EY015748 and R01EY017964).

Part I. Set-up for the ACT Assay
<ol>
	<li>Generate capped RNA for injection as suggested by the manufacturer, using their phenol:chloroform purification method (e.g. mMessage mMachine Kit; Applied Biosystems/Ambion, Austin, TX). RNA encoding a fluorescent protein is used as a tracer. We transcribe either yellow fluorescent protein (YFP) or mCherry 5 cDNA, which is in a pCS2+ expression vector.</li>
	<li>Pull borosilicate glass capillary tubes to form tapered glass tips using a micropipette puller. We use Sutter Micropipette Puller, P-97 (Sutter Instruments, Inc., Novato, CA), as previously described 6.</li>
	<li>Make sterile solutions in Table 1 from a 10 X Marc's Modified Ringer's (MMR) solution stock (10 mM MgCl2 ; 20 mM KCl; 20 mM CaCl2 ; 50 mM HEPES; 1 M NaCl; adjusted to pH 7.5, autoclaved and stored at RT).</li>
	<li>Isolate testes from a male frog and store the tissue at 4&deg;C in a sterile solution of 1X MMR/gentamicin (50 &mu;g/mL gentamicin sulfate; BioWhittaker, Cat # 17-518Z). This can be done up to a week before the start of the experiment but it is looses its efficacy over time.</li>
	<li>Egg laying solution (1X: 150 mM NaCl, 2.62 mM KCl, 0.8 mM Na2HPO4, 20 mM Trizma base, 2.62 mM NaHCO3 and 2.75 mM MgSO4; adjusted to pH 7.6) can be made as an 8X stock, which can be stored at 4&deg;C for up to two weeks. Note: watch out for contamination in this solution, as this will affect the quality of your eggs.</li>
	<li>Generate injection plates. To do this, melt 1% agarose in 0.4X MMR in a microwave, add ~6 mL melted agarose solution into 60 mm plates, gently unroll an elastomer mold (Figure 1) on top of the solution to prevent bubbles and let cool for about 20 minutes at RT. To determine the number needed, consider the number of experimental conditions (e.g. 1, YFP; 2, YFP+Noggin) and the number of transplanted host embryos needed. For our experiments, we generate at least 20 transplanted host embryos for each experimental condition. At 18-19&deg;C, embryos will be at stage 15 for about an hour. If it takes one hour to perform 20 transplants, then two fertilization time points are needed, assuming the host embryos accept all transplants. Therefore, four injection dishes will be needed (2 exp conditions x 2 fertilization time points = 4). Store the plates at 4&deg;C for up to one week.</li>
	<li>Generate cap isolation plates using 1% agarose + 0.7X MMR with molds as for the injection plate. Make the same number of cap isolation as injection plates plus one extra per fertilization time point (4 + 2 fertilization time points = 6).</li>
	<li>Inject females with 500 units human chorionic gonadotropin late in the day on the day before microinjection. Store females at 16-18&deg;C overnight.</li>
</ol>
Part II. RNA Microinjection of Xenopus laevis embryos
A. Preparation for microinjection
<ol>
	<li>Remove hormonally induced female and place them in individual tanks containing 1X egg laying solution (ELS). Note: we don't use eggs that have been in the ELS more than two hours as the quality of eggs is reduced. Therefore, about an hour before in vitro fertilization, we discard all eggs from the tank so that only freshly laid eggs are collected.</li>
	<li>Prepare the needle for microinjection by snipping off the tip to obtain a 25-35 &mu;m diameter. Place the needle onto the picoinjector and calibrate it so that it dispenses 10 nL per injection.</li>
	<li>Set an incubator to 14&deg;C.</li>
	<li>Once females have laid eggs for about an hour, remove the eggs from the tank using a transfer pipette and place them into 60 mm Petri dishes. Remove as much ELS from the eggs as possible and replace with 1X MMR. The eggs can be fertilized right away or stay in 1X MMR for up to an hour. For the transplant assay, it is best to fertilize two dishes, 80% full of a single layer of eggs in up to three separate batches at around 11 am, noon and 1 pm. After injection, they will be placed at 14&deg;C so that they will grow to the desired stages for cap isolation (stage 8.5-9) and transplantation (stage 15; see Part I, #6 above).</li>
	<li>To check the quality of the testes, we perform an early morning fertilization (~8 am), which allows us to extract testes from another male if the sperm from the first is not viable. Note: we homogenize ~0.25 testes in 1 mL 1X MMR and add approximately 5 to 7 drops per plate of oocytes.</li>
	<li>In vitro fertilize the eggs with homogenized testes and, an hour later, remove the jelly coat on the eggs using de-jelly solution (164&mu;l 1M DTT + 10mL 1M Tris, pH 8.8 up to 50 mL autoclaved nanopure water). Wait until they reach two-cell stage to begin sorting, which takes about 2 hours at 18&deg;C or 1.5 hours at room temperature (22&deg;C).</li>
	<li>Using a transfer pipette, sort uniformly pigmented and evenly dividing embryos, as shown in the left animal cap in Figure 3, into the well of an injection dish filled with 0.4X MMR + 6% Ficoll.</li>
</ol>
B. Microinjection
<ol>
	<li>Stretch Parafilm over the top of a 60 mm Petri dish lid. Pipette 2 &mu;l of your first RNA sample onto the Parafilm and aspirate using the PLI-100 to fill your injection needle. Be sure to check that the needle is still dispensing 10 nL of sample before starting injection.</li>
	<li>Inject 20-30 two-cell stage embryos (both blastomeres) with 500 pg YFP RNA in one dish and your gene of interest (e.g. 20 pg Noggin RNA) plus YFP RNA in another dish 7.</li>
	<li>Place 100-200 non-injected embryos per time point in another injection dish with the Ficoll solution. Move them in and out of the incubator at the same time as the injected embryos. These are the hosts to be used in Part IV.</li>
	<li>After injection, incubate all embryos at 14&deg;C overnight. Repeat for all time points.</li>
</ol>
Part III. Isolate animal caps from injected embryos
<ol>
	<li>Arrive early in the morning to stage the embryos. The earliest time point should be at stage 9. Remove those that did not survive. Proceed with the following steps working with each time point in batches.</li>
	<li>Pour off the Ficoll solution from each plate and add back 0.7X MMR/gentamicin solution. To a cap isolation dish, add a generous amount of 0.7X MMR/gentamicin solution. Dislodge embryos from their injection dish wells using a transfer pipette and place in the freshly prepared cap isolation dish.</li>
	<li>Isolate animal caps in 0.7X MMR + gentamicin solution, keeping 2 or 3 intact embryos for staging. Either the Gastromaster, fitted with a yellow tip bent to 400 &mu;m, or a pair of sharp number 5 forceps can be used.</li>
	<li>Put caps back in 14&deg;C incubator until the next day. The host embryos should be removed from the incubator at the same time as the caps to keep them at the same developmental stage as the donor tissue.</li>
</ol>
Part IV. Transplantation of caps onto flank
<ol>
	<li>Remove caps and embryos from the first time point from the 14&deg;C incubator and stage the embryos. Sort for uniform yellow or red fluorescent-positive caps, depending on the tracer injected (Figure 2).</li>
	<li>Count the number of caps and remove double the number of stage 12-13 embryos to a freshly prepared injection dish with 0.7X MMR/gentamicin solution. With sharp #5 forceps, remove the vitelline membrane from each. Transfer them to the plate with the caps, but be careful. Without their vitelline membranes, embryos can quickly dissociate at the water's surface.</li>
	<li>Using the gastromaster (yellow tip, 200-250&mu;m bent wire, highest setting) or #5 forceps, remove a 200 &mu;m square on the side of the embryo at stage 14-15 (Figure 3). Then, cut a cap in half (with the gastromaster or the #5 forceps) and place the tissue in the hole made in the host embryo. Rotate the embryo against the well to allow healing. Do this for as many embryos as you can while they are at stage 15. Repeat for all time points.</li>
	<li>The embryo will usually heal within 30 minutes. Remove the dead cells (white) with a gentle brushing motion with a #5 forceps. Grow them at 18&deg;C overnight.</li>
	<li>Under the dissecting fluorescence microscope, sort embryos with fluorescently-labeled, transplanted tissue. Put them back at 18&deg;C to grow until stage 42-43.</li>
	<li>Anesthetize the animals in tricaine. At this point, you can take pictures under a fluorescence dissecting microscope. After you are done, fix, section and immunostain or perform in situ hybridization using markers for your tissue of interest.</li>
</ol>
<img src="/files/ftp_upload/1932/1932fig1.jpg" alt="Figure 1" /> Figure 1. Design of Plexiglass for casting the elastomer mold used for making embryo holding dishes. A 38 x 38 mm round-cornered square (~5 mm deep) has been routed out of a piece of Plexiglass that is 50 x 50 mm square and 25 mm thick. Circular, evenly spaced wells (1.5 mm diameter) have been drilled 1 mm deep into the plastic. To make the elastomer mold for the microinjection dishes, we poured Sylgard elastomer into the Plexiglass and allowed it to solidify, as per manufacturer instructions. This elastomer mold is used to make injection and cap isolation plates.
<img src="/files/ftp_upload/1932/1932fig2.jpg" alt="Figure 2" /> Figure 2. Animal cap sorting prior to transplantation is important, as expression can vary. Animal cap on the left expresses mCherry evenly throughout tissue while the explant on the right has spotty expression and will be discarded.
<img src="/files/ftp_upload/1932/1932fig3.jpg" alt="Figure 3" /> Figure 3. A host tadpole has developed an eye-like structure on its flank from transplanted animal cap cells. A fluorescent image has been overlayed on the brightfield image.

<ol>
	<li>Smith, W. C., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin,+a+new+dorsalizing+factor+localized+to+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Lamb, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+by+the+secreted+polypeptide+noggin.">Neural induction by the secreted polypeptide noggin.</a> Science. 262, 713-718 (1993).</li><li>Green, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Methods+in+Developmental+Biology:+Xenopus+&#38;+Zebrafish.">Molecular Methods in Developmental Biology: Xenopus &#38; Zebrafish.</a> Methods in Developmental Biology. , (1999).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7, e1000174-e1000174 (2009).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J Vis Exp. , (2008).</li><li>Lan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+Elicits+Retinal+Fate+in+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate in Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. , (2009).</li></ol>

In this video, we have demonstrated our version of classic techniques used by Xenopus biologists, which we rearranged to create the animal cap transplant assay. At room temperature, Xenopus embryos develop very quickly through stages 9 and 15. By placing them at 14&deg;C, the development of the embryo is slowed down and many more transplants can be performed in one experiment. If it is necessary to grow the embryos to older stages (&gt;42/43), then we recommend using transgenic venus YFP animals, since ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>mMessage mMachine SP6 Kit</td>
<td>&nbsp;</td>
<td>Applied Biosystems/Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate Capillary Tubes (1.0mm OD x 0.5mm ID)</td>
<td>&nbsp;</td>
<td>FHC, Inc</td>
<td>27-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Sutter Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments, Inc.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin sulfate [50 mg/ml]</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BW17-528Z</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard/elastomer Kit 1.1lb</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9644388</td>
<td>&nbsp;</td>
<tr>
<td>60 x 15 mm polystyrene petri dish</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>&nbsp;</td>
<tr>
<td>human chorionic gonadotropin</td>
<td>&nbsp;</td>
<td>Intervet</td>
<td>22219</td>
<td>&nbsp;</td>
<tr>
<td>Pico-Injector Microinjection Systems</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>650003</td>
<td>&nbsp;</td>
<tr>
<td>Torrey Pines Scientific ECHOtherm Incubator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-680-21</td>
<td>&nbsp;</td>
<tr>
<td>Transfer pipette</td>
<td>&nbsp;</td>
<td>Krackeler</td>
<td>6290-20635A</td>
<td>&nbsp;</td>
<tr>
<td>Dumostar #5 Forceps</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11295-10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

tissue determination using the animal cap transplant (act) assay in xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Watch this Scientific Journal Video about Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis at JoVE.com

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

tissue-determination-using-animal-cap-transplant-act-assay-xenopus

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12.3K visualizações.  SUNY Upstate Medical University. Superexpressão de genes animais tampas de produto (s) são transplantadas para o flanco de desenvolver Xenopus laevis Embriões, a fim de estabelecer se o tecido é determinada.

Vídeo: Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...