The authors would like to acknowledge the cooperation and financial contributions of the following research funding agency: Alberta Livestock and Meat Agency Ltd., Pork CRC, Alberta Pork, Hypor A Hendrix Genetics Company and NSERC CRSNG.

In accordance with the Canadian Council on Animal Care guidelines and with approval of the Faculty Animal Policy and Welfare Committee - Livestock of the University of Alberta, pregnant sows were euthanized by trained staffs at approximately Day 30 of pregnancy and embryos were collected. Use examination gloves at all times during the procedures.
1. Sample Collection and Sample Storage
<ol>
	<li>Euthanize sow using captive bolt followed by exsanguination. Wear examination gloves to collect the reproductive tracts and embryos from each euthanized sow. 
	Note: Rapid collection of samples and transfer embryos to dry ice or liquid nitrogen will result in higher quality tissues for other molecular works such as microarray and qPCR.</li>
	<li>Dissect the uterus and gently separate all the embryos within their extraembryonic placental membranes from the underlying uterine wall21. Make sure there is no maternal tissue contamination.</li>
	<li>Record length and weight of all viable embryos prior to freezing.</li>
	<li>Collect each individual embryo and immediately wrap it up in an aluminum foil before snap freezing with liquid nitrogen.</li>
	<li>Transfer the frozen embryos from liquid nitrogen directly to a -80 &#176;C freezer.</li>
</ol>
2. Grinding Embryos
<ol>
	<li>Label all sample tubes (15 ml) with the sample ID needed for the number of samples to be analyzed.</li>
	<li>Fill thermal container with dry ice to half full and insert the pre-labelled sample tube in the dry ice.</li>
	<li>Wear winter gloves with another pair of examination gloves on top for transferring the pre-chilled mortars and pestles from -80 &#176;C freezer to dry ice container.</li>
	<li>Place the frozen embryo inside the mortar on the dry ice.</li>
	<li>Pour adequate liquid nitrogen to cover the embryo and grind it with the pestle into a fine powder.</li>
	<li>Transfer the embryo powder to a pre-labeled sample tube with a microspatula (Figure 1) and place the tube in a -80 &#176;C freezer.</li>
	<li>Use a clean pre-chilled mortar and pestle for each embryo and change the examination gloves after grinding each embryo to avoid cross contamination between samples.</li>
</ol>
3. Genomic DNA Preparation using Modified Sodium Hydroxide Method
<ol>
	<li>Label all the microcentrifuge tubes (1.5 ml) needed for the number of samples to be analyzed.</li>
	<li>Transfer the sample tubes containing embryo powder from the -80 &#176;C freezer to a container with dry ice prior to starting.</li>
	<li>Pipette 180 &#181;l of 50 mM NaOH (sodium hydroxide) into each pre-labelled microcentrifuge tube.</li>
	<li>Turn on an incubator and pre-heat to 95 &#176;C.</li>
	<li>Use a toothpick to transfer the correct amount of embryo powder (about 5-10 mg) (Figure 2) from the sample tube into a pre-labelled microcentrifuge tube containing the 50 mM NaOH solution.</li>
	<li>Pull up the same toothpick slowly to visualize the DNA lysate as a sticky, gooey, white transparent-like substance (Figure 3).</li>
	<li>Transfer the microcentrifuge tubes with DNA lysate to the pre-heated incubator at 95 &#176;C for five minutes. Immediately transfer the tube to a styrofoam box filled with ice.</li>
	<li>Add 20 &#181;l of 1 M Tris-HCl directly into the microcentrifuge tube and mix it by gently tapping the tube. Use a pH paper to ensure the pH is approximately 8.0 (Figure 4) prior to centrifugation. 
	Note: When dealing with a large number of sample preparations, it is acceptable to randomly pick few samples to check the pH.</li>
	<li>Centrifuge the tube with the DNA lysate at 2,000 x g in a microcentrifuge for two min at room temperature to remove undissolved tissue debris.</li>
	<li>Transfer 150 &#181;l of the top clear supernatant into a new tube or 96 well plates. 
	Note: The clear DNA lysate is ready to use as a template in PCR reaction and it is stable at 4 &#176;C for two weeks, or it can be kept at -20 &#176;C for a year.</li>
</ol>
4. Design Sex-specific PCR Primers
<ol>
	<li>Obtain accession numbers for Porcine sex determining region Y (SRY) (NM_214452.3) and and zinc finger protein X-linked (ZFX) genes (XM_005673501.1) from NCBI website http://www.ncbi.nlm.nih.gov/.</li>
	<li>Copy and paste these NCBI accession numbers to an online primer design tool. 
	Note: The best primers information including length, Tm and GC% as well as PCR product size (bp) will be generated on the computer screen (Figure 5).</li>
	<li>Validate the specificity of these primers using nucleotide Blast program (Blastn) against the current porcine genomic database 104 to ensure these sequences are only located on X and Y chromosome for SRY and ZFX respectively (Figure 6).</li>
</ol>
5. Genomic DNA PCR Directly from DNA Lysates
<ol>
	<li>Use only 1 &#181;l of the DNA lysate as a template for a 15 &#181;l PCR reaction. 
	Note: A high throughput screening method for 96-well plates PCR can be performed in similar way using a multichannel pipette.</li>
	<li>Do the following just for the first PCR: prepare one PCR tube for the no template negative control and two additional tubes for positive controls by adding 1 &#181;l (0.5 ng) of commercially obtained known sex porcine genomic DNA. Later, include a sample from the last successful sexing analysis as a positive control and run together with the new PCR reaction for sex determination.</li>
	<li>Use any HotStart ready mix PCR enzyme and prepare a master mix by adding primers and nuclease free water depending on the total number of PCR reactions. Add 1 &#181;l of the DNA lysate into a pre-existing PCR tube with 14 &#181;l of the PCR master mix. Add primers such that the final concentration of the primers from two sex-specific genes is 0.3 &#181;M in total of 15 &#181;l PCR reaction.</li>
	<li>Set up the following PCR program in a thermal cycler: 95 &#176;C for 3 min, 35 cycles each with a 20 sec melting step 98 &#176;C, followed by a 15 sec annealing step at 65 &#176;C, followed by a 15 sec elongation step at 72 &#176;C. In a final step, incubate the reactions for 1 min at 72 &#176;C and continue to incubate at 4 &#176;C until removal of the PCR tube for gel electrophoresis verification to determine the sex. 
	Note: PCR conditions and optimization are according to the manual of the PCR kit mentioned in the Materials Table.</li>
	<li>Add the appropriate amount of non-toxic green- fluorescent SYBR DNA gel stain when preparing a 2% TBE agarose gel. 
	Note: Ethidium bromide can be substituted for green- fluorescent stain if it is not available.</li>
	<li>Add 1.5 &#181;l of the loading dye (10x) into the PCR tube and mix well by pipetting the PCR reaction buffer up-and-down.</li>
	<li>Load 10 &#181;l of the sample into the well and run the agarose gel with appropriate voltage settings (small gel apparatus at 100 V, 96-well gel apparatus at 150 V until the dye band runs half way through the gel).</li>
	<li>Observe and adjust the bands intensities under the fluorescent light setting using a laser scanner and capture an image of the gel. Note: Common gel imager can be substituted for the ethidium bromide gel.</li>
	<li>Determine the sex of the porcine embryos by identifying embryos with one band as a female and two bands as a male (Figure 7).</li>
</ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+diet+and+other+factors+affecting+offspring+sex+ratio:+a+review.">Maternal diet and other factors affecting offspring sex ratio: a review.</a> Biol Reprod. 71, 1063-1070 (2004).</li><li>Gorecki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+ratio+in+litters+of+domestic+pigs+(Sus+scrofa+f.+domestica+Linnaeus,+1758).">Sex ratio in litters of domestic pigs (Sus scrofa f. domestica Linnaeus, 1758).</a> Biol. Lett. 40, 111-118 (2003).</li><li>Chen, Z. Y., Dziuk, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+initial+length+of+uterus+per+embryo+and+gestation+stage+on+prenatal+survival,+development,+and+sex+ratio+in+the+pig.">Influence of initial length of uterus per embryo and gestation stage on prenatal survival, development, and sex ratio in the pig.</a> J. Anim. Sci. 71, 1895-1901 (1993).</li><li>Vinsky, M. D., Novak, S., Dixon, W. T., Dyck, M. K., Foxcroft, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+restriction+in+lactating+primiparous+sows+selectively+affects+female+embryo+survival+and+overall+litter.">Nutritional restriction in lactating primiparous sows selectively affects female embryo survival and overall litter.</a> Reprod. Fertil. Dev. 18, 347-355 (2006).</li><li>Cassar, G., King, W. A., King, G. 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The authors declare that they have no competing financial interests.

Most of the existing protocols related to porcine embryos DNA sex typing are only suitable for early stage pre-implantation stage15,16. We have successfully developed a protocol suitable for porcine embryo screening during late gestation. Based on studies with similar developmental stages of embryos from previous studies6,18, the present protocol is considered to be safer and low cost.
This protocol is also suitable for a large number of samples for sex typing using PCR b...

The domestic pig has become a fundamental research subject in development, genetics and nutrition in both human and livestock sectors. The potential of pigs as biomedical models for human research can be attributed to their physiological similarities. In livestock, the manipulation of sex ratio can enhance the effectiveness of selection and genetic improvement programs1. Sexing individual embryos is a fundamental tool used in many experimental investigations including but not limited to genotype, epigenetics and X inactivation of sexual dimorphism during early embryo development2.
Studies in mice suggest that maternal diet and other factors may result in gender imbalance3. In pigs, causes of sex ratio imbalance include paternal breed4, uterine capacity5, and the sow&#39;s metabolic condition6. Since the differences observed in embryos and litters can be influenced by sexual dimorphism, researchers should be aware of embryo sex and sex ratios before drawing conclusions regarding their research. The development of efficient tools and protocols for sexing pig embryos at Day 30 of development will be discussed here.
Various methods of sex typing have been developed for genetic studies in model organisms and livestock. Particularly in livestock, identifying male and female early embryos is a very common practice to enhance genetic selection for breeding programs. Early embryos karyotyping in pig using Giemsa7 or the intense fluorescence8,9 techniques have been used for sex typing. However, these methods are time consuming and not suitable for screening large numbers of embryos quickly and accurately.
The most effective sex typing method is DNA amplification using a heat stable DNA polymerase and a pair of primers. DNA sexing by PCR method is more specific, rapid and sensitive, only requiring a minute amount of cellular materials. The first PCR-based embryo sexing was performed on humans10, and later in mice11, cattle12, buffalo13 and sheep14 pre-implantation embryos. In the pig, the earliest DNA sex typing method was established for pre-implantation embryos via a single pair of Y-chromosome specific DNA primers15. However, the most common PCR primers for sex determination were selected from the Y-chromosome of male specific SRY gene16 and the non-sex discriminative region of a zinc-finger gene located at both X and Y chromosome17. Subsequently, these primers have been applied to determine the sex of Day 30 embryos in this study with improved specificity of the primers to detect only the X- chromosome of a zinc-finger gene.
Genomic DNA from porcine pre-implantation embryos can be extracted by exposing an intact blastocyst to buffer with proteinase K16 or by taking a biopsy of a few cells from the individual early cleavage embryos15 and using them for direct PCR. However, release of DNA from porcine blood, hair, tissues or a large conceptus over a few centimeters in size is not effectively done using the proteinase K method. DNA extraction methods for these materials have been established using either time consuming phenol/chloroform protocols6 or expensive column based kits18. In order to avoid the use of potentially toxic chemicals, there is a trend to develop inexpensive, easy and phenol-free DNA extraction methods. This type of protocol for the isolation of PCR-quality genomic DNA from mouse19 and zebrafish20 tissues has been established using hot sodium hydroxide and Tris (HotSHOT). This study provides a protocol to obtain DNA with modified HotSHOT and redesigned duplex primer pairs for PCR sex typing directly from cell lysates of Day 30 porcine embryos with high accuracy.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">KAPA HiFi HotStart Ready Mix PCR kit&#160;</td>
			<td style="width:203px;">KAPABiosystems</td>
			<td style="width:189px;">KR0370</td>
			<td style="width:463px;">other Hot Start Taq polymerase can be used after optimization</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:189px;">S33102</td>
			<td>Ethidium bromide can be substituted for SYBR Safe</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Pig female and male genomic DNA</td>
			<td style="width:203px;">Zyagen</td>
			<td style="width:189px;">GP-160-F1 &#38; GP-160-M5</td>
			<td>Postive controls from the tissues of known sex DNA can be used.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Typhoon FLA 9500 laser scanner</td>
			<td style="width:203px;">GE Healthcare Life Sciences</td>
			<td style="width:189px;">28-9969-43</td>
			<td>other imaging system can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Free Soft Nitrile Examination Gloves</td>
			<td style="width:203px;">WWR</td>
			<td>89038-270</td>
			<td>any other examination glove can be used</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Sodium hydroxide, solid&#160;</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">BP 359 -212</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">0030 108.051</td>
			<td>heat resistant</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">ThermoStat plus</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22670204</td>
			<td>Use as a incubator for 95C, don&#39;t need to use the heater</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Toothpick</td>
			<td>Bunzl Plc</td>
			<td style="width:189px;">75200815</td>
			<td style="width:463px;">Any round wooden toothpicks can be used - quality wood</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:331px;">Microcentrifuge 5417R/5417C</td>
			<td style="width:203px;">Eppendorff</td>
			<td style="width:189px;">22621807</td>
			<td>This model was discontinued. But another newer model can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microspatula</td>
			<td style="width:203px;">Fisher</td>
			<td style="width:189px;">SDI28540115</td>
			<td>Autoclaved before use each time.</td>
		</tr>
	</tbody>
</table>

accurate and phenol free dna sexing of day 30 porcine embryos by pcr

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

A representative result of sex determination from 345 DNA lysates screening by PCR is shown in Figure 7 and summarized in Table 1.
As can be seen in Figure 7, the primers annealing temperature at 65 &#176;C is the optimal condition in this PCR protocol generating similar intensity and predicated amplicon sizes (Figure 5) among different samples.
<p class="jo...

Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

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9.9K Views.  University of Alberta. This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

Video: Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...