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>

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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 by transferring of the DNA lysates into a 96-well plate. The plate format allows high-throughput screening by allowing the transfer 1 &#181;l of DNA lysate for the PCR reaction using a multichannel pipette.
However, several steps involved in the procedure should be done carefully. In order to perform the modified 50 mM NaOH lysis method more efficiently, the individually frozen embryo has to be grounded into fine powder using a pre-chilled (-80 &#176;C) mortar and pestle. Avoid spilling embryo powder on the dry ice, by gently and slowly scraping the powder into the 15 ml tube, as shown in Figure 1. Changing examination gloves after each sample is highly recommended to avoid powder on the glove transferring to another embryo sample.
The appropriate amount of embryo powder to be added to the modified 50 mM NaOH lysis solution is shown in Figure 2B. An excessive amount of embryo powder is presented in Figure 2A and should be avoided. Likewise, in Figure 2C, the amount of embryo powder could be insufficient for downstream procedures. Although weighing the embryo powder for each sample is possible, it is not practical when screening embryo sex in a large scale study involving over a hundred embryos.
The amount of DNA may be visibly verified using a toothpick to check for the sticky gooey-like substance as shown in Figure 3, if it is doubtful whether the appropriate amount of powder was added in the previous step.
Adding the right amount of neutralization solution is important after lysing the embryo powder. Figure 4 shows the pH change after and before adding neutralization solution. This step is important to make sure that the pH is slightly alkaline (around 8.0) and therefore similar to most PCR reaction buffers.
Specificity of the oligo primers is the most important element for the accuracy of DNA sexing during PCR. The specificity of the oligo primers can be confirmed through the NCBI Blastn program. A single location will be detected on the X chromosome at the 3&#39; non-coding region of the ZFX gene for each primer sequence (Figure 6A) for females. A similar situation is detected only on Y chromosome at the coding region of SRY gene (Figure 6B) for males.
Error rate due to no sex identification could be very low (&#60;1%) as shown in Table 1, when all steps proceed carefully and correctly. However, in some rare case, cross contamination between DNA lysates of two different samples is possible and can be detected by PCR as indicated with a red star in the gel (Figure 7). This type of banding pattern in female can be interpreted as cross contamination with a male sample.
It is unlikely that the appearance of a very faint lower band is due to the primer competition for PCR reagents. In this case the smaller amplicon (400 bp) related to the SRY gene will generate more PCR product faster than the higher band corresponding to the ZFX gene. In this protocol, one disadvantage is the determination of cross contamination of a male sample with a female. Because our main goal is not quantification of copy number for each target gene, a tiny amount of powder from the female sample would not be easy to visualize on the gel.
The DNA sexing protocol presented here is the most cost effective method to identify embryo sex during late gestation and can be applied to others large livestock animals such as cattle, goat and sheep. Also, similar DNA sexing method has been applied in pigs related to sows metabolic condition18. Another research investigation using DNA sexing could be applied to study the genomic imprinting mechanism during prenatal programming of postnatal development in the pig22. A wide range of applications in livestock using DNA PCR sexing is possible such as pre-sex selection breeding programs, and epigenetic effects of nutrition and infectious diseases.

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><tbody><tr><td>KAPA HiFi HotStart Ready Mix PCR kit&amp;nbsp;</td><td>KAPABiosystems</td><td>KR0370</td><td>other Hot Start Taq polymerase can be used after optimization</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Life Technologies</td><td>S33102</td><td>Ethidium bromide can be substituted for SYBR Safe</td></tr><tr><td>Pig female and male genomic DNA</td><td>Zyagen</td><td>GP-160-F1 &amp;amp; GP-160-M5</td><td>Postive controls from the tissues of known sex DNA can be used.</td></tr><tr><td>Typhoon FLA 9500 laser scanner</td><td>GE Healthcare Life Sciences</td><td>28-9969-43</td><td>other imaging system can be used</td></tr><tr><td>Free Soft Nitrile Examination Gloves</td><td>WWR</td><td>89038-270</td><td>any other examination glove can be used</td></tr><tr><td>Sodium hydroxide, solid&amp;nbsp;</td><td>Fisher</td><td>BP 359 -212</td><td>Molecular Biology Grade</td></tr><tr><td>Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td><td>Eppendorff</td><td>0030 108.051</td><td>heat resistant</td></tr><tr><td>ThermoStat plus</td><td>Eppendorff</td><td>22670204</td><td>Use as a incubator for 95 &amp;#176;C , don&#039;t need to use the heater</td></tr><tr><td>Toothpick</td><td>Bunzl Plc</td><td>75200815</td><td>Any round wooden toothpicks can be used - quality wood</td></tr><tr><td>Microcentrifuge 5417R/5417C</td><td>Eppendorff</td><td>22621807</td><td>This model was discontinued. But another newer model can be used</td></tr><tr><td>Microspatula</td><td>Fisher</td><td>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.
Two amplified products of SRY (400 bp) and ZFX (506 bp) are shown in the gel picture (Figure 7). Male embryos showed two DNA fragments with the correct size of the upper (400 bp) and lower (506 bp) bands. The intensity of these two bands was shown to be equal in most male individuals. All the female embryos only showed a single DNA fragment corresponding to the ZFX gene located in the X-chromosome.
DNA lysates obtained from the modified 50 mM NaOH can be used directly for PCR with a high success rate and accuracy. DNA lysates did not show any PCR reaction inhibition after screening 345 embryos. Only three individuals were not sexed and the percentage of female embryos was slightly higher than males (Table 1).
<img alt="Figure 1" src="/files/ftp_upload/53301/53301fig1.jpg" /> 
Figure 1: Embryo Powder Transfer. Transferring embryo powder to a 15 ml tube.
<img alt="Figure 2" src="/files/ftp_upload/53301/53301fig2.jpg" /> 
Figure 2: Collecting and Transferring Embryo Powder with a Toothpick. (A) An excessive amount of embryo powder adhering to the toothpick. (B) Correct amount of embryo powder adhering to the toothpick to be transferred to the alkaline lysis reagent solution. (C) An insufficient amount of embryo powder adhering to the toothpick.
<img alt="Figure 3" src="/files/ftp_upload/53301/53301fig3.jpg" /> 
Figure 3: DNA Visualization Technique. Slowly pull up the toothpick, after adding the embryo powder, to determine if the embryo DNA has dissolved as a sticky gooey white transparent-like substance.
<img alt="Figure 4" src="/files/ftp_upload/53301/53301fig4.jpg" /> 
Figure 4: pH Confirmation. (A) pH of the sodium hydroxide (alkaline lysis reagent) solution is 12.0. (B) After addition neutralization buffer to the lysis buffer, color indication of pH paper is green and the pH should be around 8.0.
<img alt="Figure 5" src="/files/ftp_upload/53301/53301fig5.jpg" /> 
Figure 5: Sex-specific Primer Information. Sequence names, Sequences and the amplicon length obtained from the primer design tool. <a href="https://www.jove.com/files/ftp_upload/53301/53301fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53301/53301fig6.jpg" /> 
Figure 6: Mapping of Porcine Sex-specific Primers on Chromosome X and Y. (A) Location of the forward and reversed primers specified on the ZFX gene. (B) Location of the forward and reversed primers specified on the SRY gene. <a href="https://www.jove.com/files/ftp_upload/53301/53301fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53301/53301fig7.jpg" /> 
Figure 7: PCR Amplification of Porcine Sex-specific Genes from Day 30 Embryos. 2% TBE agarose gel stained with SYBR DNA gel stain with the known positive controls are indicated with male and female symbols. Two bands indicated as males and a single band as females. Red star indicated the possible sample contamination in the well with the appearance of a very faint lower band (400 bp) compared to upper band (506 bp) PCR product caused by SRY Y-specific primers. Red arrow indicates the no template PCR negative control. <a href="https://www.jove.com/files/ftp_upload/53301/53301fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Sexing PCR</td>
			<td>Embryo Count</td>
			<td>%</td>
		</tr>
		<tr>
			<td>&#9792;</td>
			<td>185</td>
			<td>53.60%</td>
		</tr>
		<tr>
			<td>&#9794;</td>
			<td>157</td>
			<td>45.50%</td>
		</tr>
		<tr>
			<td>Unknown</td>
			<td>3</td>
			<td>0.90%</td>
		</tr>
		<tr>
			<td>Total&#160;</td>
			<td>345</td>
			<td>100%</td>
		</tr>
	</tbody>
</table>
Table 1: Percentage of Female, Male and Unknown after PCR Sexing among 345 Day 30 Embryos.

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, ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.0K 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

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Genetics

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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

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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis