Name: cryopreservation of mouse embryos by ethylene glycol-based vitrification
Uploaded: 2011-11-18T12:00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification at JoVE.com

This study was conducted in cooperation with the National BioResource Project, the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The overall scheme of the experiment is shown in Fig. 1.
1. Reagent Preparation
<ol>
 <li>Make the base medium (here in known as PB1) in a 100 ml glass bottle according to the following chart below:
 <table border="1">
 <tbody>
 <tr>
<td>&nbsp;</td>
 <td>M.W.</td>
 <td>mM</td>
 <td>mg/100ml</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>58.4</td>
 <td>136.98</td>
 <td>800.0</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>74.6</td>
 <td>2.68</td>
 <td>20.0</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>136.1</td>
 <td>1.47</td>
 <td>20.0</td>
 </tr>
 <tr>
 <td>Na2HPO4.12H2O</td>
 <td>358.14</td>
 <td>8.04</td>
 <td>288.1</td>
 </tr>
 <tr>
 <td>MgCl2,6H2O</td>
 <td>203.3</td>
 <td>0.49</td>
 <td>10.0</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>180.2</td>
 <td>5.56</td>
 <td>100.0</td>
 </tr>
 <tr>
 <td>Na pyruvate</td>
 <td>110</td>
 <td>0.33</td>
 <td>3.6</td>
 </tr>
 <tr>
 <td>CaCl2,2H2O</td>
 <td>147</td>
 <td>0.9</td>
 <td>13.2</td>
 </tr>
 <tr>
 <td>Penicillin G</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>6.0 (approx)</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Make the Ficoll-sucrose (FS) solution :
 <ol>
 <li>Add the following chemicals to 14 ml of PB1 solution in a 50 ml tube.
 <table border="1">
 <tbody>
 <tr>
 <td>Ficoll 70</td>
 <td>6.0 g</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>3.424 g</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>42.0 mg</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Mix Ficoll 70 and sucrose in PB1 solution and shake well until completely dissolved.</li>
 <li>After checking complete dissolution above, add BSA on the surface of the solution and keep it at 4oC until BSA is completely dissolved (&gt; 4 hours or overnight).</li>
 </ol>
 </li>
 <li>Make the equilibration solution (EFS20) and the vitrification solution (EFS40) in a 50 ml tube:
 <ul>
 <li>EFS20: 20%(v/v) ethylene glycol, 24% (w/v) Ficoll, and 0.4 mol/L sucrose in PB1 with BSA</li>
 <li>EFS40: 40%(v/v) ethylene glycol, 18% (w/v) Ficoll, and 0.3 mol/L* sucrose in PB1 with BSA 
 *For embryos from the BALB/c or ICR strain, increase the concentration of sucrose to 0.9 mol/L sucrose because they are more sensitive to cryodamage6.</li>
 </ul>
 <table border="1">
 <tbody>
 <tr>
<td>&nbsp;</td> 
<td>EFS20 solution</td>
 <td>EFS40 solution</td>
 </tr>
 <tr>
 <td>Ethylene glycol</td>
 <td>1 ml</td>
 <td>2 ml</td>
 </tr>
 <tr>
 <td>FS solution</td>
 <td>4 ml</td>
 <td>3 ml</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Sterilize the solution by filtration using a 0.45 &mu;m filter. Make aliquots in 5 ml polystyrene tubes and store at 4&deg;C. They can be used for about 6 months.</li>
 <li>Preparation of embryo thawing solution containing 0.75 M sucrose (TS1)
 <ol>
 <li>Dissolve 7.7 g of sucrose in PB1 (prepared in step 1.1) and bring the total volume to 30 ml. Mix by gentle shaking until sucrose is completely dissolved.</li>
 <li>Add 90 mg of BSA onto the surface the solution and leave it stand until completely dissolved.</li>
 <li>Sterilize the solution by filtration.</li>
 <li>Aliquot and store at 4&deg;C. They can be used for about 1 month.</li>
 </ol>
 
 Note: TS1 can also be prepared from commercially available M2. 
 <ol start="6">
 <li>Dissolve 7.7 g of sucrose in M2 and bring the total volume to 30 ml.</li>
 <li>Mix by gentle shaking until sucrose is completely dissolved.</li>
 <li>Sterilize the solution by filtration.</li>
 <li>Aliquot and store at 4oC. They can be used for about 1 month.</li>
 </ol>
 </li>
 <li>TS2 (thawing solution containing 0.25 M sucrose):
 <ol>
 <li>Dilute 10 ml TS1 with 20 ml volume of PB1 or M2, as appropriate.</li>
 <li>Aliquot and store at 4&deg;C. They can be used for about 1 month.</li>
 </ol>
 </li>
</ol>
2. Vitrification of 2-cell Mouse Embryos
<ol>
 <li>Prepare 2-cell mouse embryos by natural mating or conventional in vitro fertilization techniques.</li>
 <li>Aliquot 50 &mu;l EFS40 into a cryotube. Hereafter, perform the rest of the vitrification procedure at the room temperature.</li>
 <li>Aliquot 50 &mu;l of EFS20 on the bottom of a 35 mm or 60 mm plastic Petri dish.</li>
 <li>Transfer up to 30 embryos to EFS20 using a glass capillary with only the minimum amount of the culture medium. Start a timer for 2 min. 
 Note: Place embryos on the bottom of the drop. This makes efficient dehydration of embryos. Check the morphology of embryos by a stereomicroscope. Dehydrated embryos show a shrunken morphology, as shown in Fig. 2A. If they are not dehydrated enough, wait for more 1 or 2 min.</li>
 <li>At about 1.5 min, pick up the embryos from the EFS20 drop with only the minimum amount of the solution. Transfer them to EFS40 in the cryotube prepared in step 2.1. Note: For the best results, adjust the timing of picking up embryos so that all embryos can be transferred into EFS40 at around 2 min.</li>
 <li>Wait for 1 min.</li>
 <li>Put the cryotube directly into liquid nitrogen (LN2).</li>
</ol>
3. Thawing Vitrified 2-cell Mouse Embryos
<ol>
 <li>Before thawing, prepare a dish with 10 &mu;l drops of embryo culture medium for recovered embryos (see step 3.13). Cover the dish with oil and place in a CO2 incubator until use. Note: Although any media for routine embryo culture may be used in this experiment, we recommend media with higher osmolarity such as M16 medium.</li>
 <li>Warm TS1 to 37&deg;C.</li>
 <li>Put on a face mask and cryogloves. Open the LN2 tank and retrieve a cryotube containing embryos.</li>
 <li>Quickly open the top of the tube and discard LN2. Wait for 30 sec to prevent TS1 from freezing at the next step.</li>
 <li>Use a 1000ul pipette to add 850 &mu;l of TS1 (37&deg;C) into the tube and mix the solution by gentle pipettings (about ten times in 25 seconds) until the solution is evenly dissolved.</li>
 <li>Transfer the entire volume of the tube to a plastic 60 mm Petri dish (or a watch glass) (Fig. 3A). Note: Embryos should be handled at room temperature (22-25&deg;C) until they are placed in a CO2 incubator at Step 3.14.</li>
 <li>Start a timer for 3 min.</li>
 <li>At around 2 min in TS1, gently shake the dish until the medium containing embryos is spread over the surface of the dish (Fig. 3B). Note: This will help the embryos go down to the bottom because they are floating near the surface when transferred to TS1.</li>
 <li>Put three 50 &mu;l drops of TS2 onto the dish (Fig. 3C).</li>
 <li>After 3 min in TS1, check the morphology of embryos by a stereomicroscope; they should be slightly shrunken. See the morphology of embryos in earlier (Fig. 2B) and later (Fig. 2C) in TS1. Note: If the embryos are still swollen, keep them in TS1 for more 1 to 3 min.</li>
 <li>Pick up the embryos and transfer them to the first drop of TS2 (Fig. 3D). Start a timer for 3 min</li>
 <li>After 3 min, transfer embryos to the second drop and then to the third drop (Fig. 3E).</li>
 <li>Transfer the embryos to the culture medium prepared at Step 3.1. The embryos are in the shape shown in Fig. 2D.</li>
 <li>Place dish in a CO2 incubator. About 10 min later, transfer embryos to the next drop of culture medium to wash out sucrose that has been carried over from TS2.</li>
 <li>Continue culture in a CO2 incubator until embryo transfer. 
 Note: Transfer embryos to the oviducts of recipient females on the day of thawing. Long culture in vitro may decrease the viability of embryos in some strains of mice.</li>
</ol>
4. Representative Results:
In vitro- and in vivo-development of embryos after thawing is presented in Tables 1 and 2. The advantages of this protocol are the high survivability of embryos after thawing and its broad applicability to different strains of mice.
<table border="1">
 <tbody>
 <tr>
 <td>Strain</td>
 <td>Total No. of tubes</td>
 <td>No. of embryos vitrified</td>
<td>Recovered (No. (%))</td>
<td>Morphologically normal (No. (%))</td>
<td>Development to blastocysts (No. (%))</td>
 </tr>
 <tr>
 <td>C57BL/6J</td>
 <td>20</td>
 <td>400</td>
 <td>397 (99)</td>
 <td>394 (99)</td>
 <td>342 (87)</td>
 </tr>
 <tr>
 <td>BALB/cA</td>
 <td>15</td>
 <td>300</td>
 <td>296 (99)</td>
 <td>282 (95)</td>
 <td>238 (84)</td>
 </tr>
 <tr>
 <td>ICR</td>
 <td>24</td>
 <td>480</td>
 <td>474 (99)</td>
 <td>443 (93)</td>
 <td>398 (90)</td>
 </tr>
 </tbody>
</table>
Table 1. In vitro-development of vitrified-thawed embryos in common mouse strains
<table border="1">
 <tbody>
 <tr>
 <td>Condition of embryos</td>
 <td>No. of recipient females</td>
 <td>No. of embryos transferred</td>
 <td>Implantation sites (No. (%))</td>
 <td>Live offspring (No. (%))</td>
 </tr>
 <tr>
 <td>Fresh</td>
 <td>12</td>
 <td>180</td>
 <td>141 (78.3)</td>
 <td>110 (61.1)</td>
 </tr>
 <tr>
 <td>Vitrified</td>
 <td>16</td>
 <td>242</td>
 <td>202 (83.5)</td>
 <td>125 (51.7)</td>
 </tr>
 </tbody>
</table>
Table 2. In vivo-development of vitrified-thawed embryos in C57BL/6J mice.
<img alt="Figure 1" src="/files/ftp_upload/3155/3155fig1.jpg" /> 
Figure 1. The overall scheme of the experiment including equilibration, vitrification, and thawing of embryos.
<img alt="Figure 2" src="/files/ftp_upload/3155/3155fig2.jpg" /> 
Figure 2. The morphology of embryos at each step of thawing.
<img alt="Figure 3" src="/files/ftp_upload/3155/3155fig3.jpg" /> 
Figure 3. Thawing procedure of embryos. All procedures are performed at room temperature. (A), Add 850 &mu;l of TS1 (37&deg;C) into a cryotube and transfer the entire volume of the solution in the cryotube onto a plastic dish. At this time, the embryos look swollen as shown in Fig. 2B. (B), Spread the solution over the surface of the dish by gentle shaking. (C), Place three 50 &mu;l drops of TS2 on the plastic dish. (D), Transfer the embryos to the first drop of TS2. After 3 min, the embryos look shurunken as shown in Fig. 2C. (E), Transfer them serially into the remaining TS2 drops and then to the culture medium.

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196&#176;+and+-269&#176;C.">Survival of mouse embryos frozen to -196&#176; and -269&#176;C.</a> Science. 187, 411-414 (1972).</li><li>Rall, W. F., Fahy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-free+cryopreservation+of+mouse+embryos+at+-196&#176;C+by+vitrification.">Ice-free cryopreservation of mouse embryos at -196&#176;C by vitrification.</a> Nature. 313, 573-575 (1985).</li><li>Yoshiki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+resources+at+the+RIKEN+BioResource+center.">The mouse resources at the RIKEN BioResource center.</a> Exp. Anim. 58, 85-96 (2009).</li><li>Mochida, K., Ogura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+embryos+in+laboratory+species.">Cryopreservation of embryos in laboratory species.</a> J. Mamm. Ova. Res. 27, 87-92 (2010).</li><li>Kasai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+mouse+embryos+cryopreservation+in+a+low+toxicity+vitrification+solution,+without+appreciable+loss+of+viability.">A simple method for mouse embryos cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability.</a> J. Reprod. Fert. 89, 91-97 (1990).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing+methods.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Kasai, M., Mukaida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+animal+and+human+embryos+by+vitrification.">Cryopreservation of animal and human embryos by vitrification.</a> Reprod. Biomed. Online. 9, 164-170 (2004).</li><li>Miyake, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+mouse+oocytes+and+embryos+at+various+stages+of+development+in+an+ethylene+glycol-based+solution+by+a+simple+method.">Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method.</a> Theriogenology. 40, 121-134 (1993).</li><li>Han, M. S., Niwa, K., Kasai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+of+rat+embryos+at+various+developmental+stages.">Vitrification of rat embryos at various developmental stages.</a> Theriogenology. 59, 1851-1863 (2003).</li><li>Jin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+vitrification+of+mouse+embryos.">Equilibrium vitrification of mouse embryos.</a> Biol. Reprod. 82, 444-450 (2010).</li></ol>

We have nothing to disclose.

Since the first report of mouse embryo vitrification by Rall and Fahy in 19852, several technical improvements have been made to increase the survivability of embryos after thawing. One of the most successful modifications was achieved by use of ethylene glycol as a cryoprotectant because of its low toxicity and high membrane-permeability. Such advantages enable us to handle freezing embryos at room temperature4; other vitrification methods require embryo handing at cooler temperatures and are not always applicable to some strains of mice including BALB/c6. The first ethylene glycol-based vitrification was developed by Kasai et al. in 19905,7. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. Therefore, the vitrification method described here may be applicable to many laboratories using mice as research models. The same method can also be used for mouse embryos at the morula and blastocyst stages8 and rat embryos9. However, we have recently found that the quality of cryotubes may affect the survival rates of embryos after freezing-thawing. Thus, it is essential to examine the inside surface of the cryotubes for its smoothness before vitrifying embryos (e.g.; see at Table of specific reagents and equipment). 
Vitrification methods have many advantages over conventional slow freezing methods, but they inherently have a disadvantage in respect to transportation purpose. As vitrified embryos should be kept at below -120&deg;C to maintain their viability, dry shippers are generally used for their safe transportation. Dry shippers are heavy and bulky, and their round-trip is expensive, especially for international transportation. We are now currently developing a new vitrification method by which vitrified embryos can be stored at dry-ice temperature (about -80&deg;C) for at least 7 days10. This method should be the vitrification of the next generation.

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments (optional)</td>
</tr>
<tr>
<td>Bovine serum albumin</td>
<td>Merck Biosciences (Calbiochem)</td>
<td>12657</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ethylene glycol</td>
<td>Wako Pure Chemical Industries</td>
<td>058-00986</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ficoll 70</td>
<td>GE Healthcare</td>
<td>17-0310-10</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Glucose</td>
<td>Wako Pure Chemical Industries</td>
<td>041-00595</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NaCl</td>
<td>Wako Pure Chemical Industries</td>
<td>191-01665</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>KCl</td>
<td>Wako Pure Chemical Industries</td>
<td>163-03545</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>KH2PO4</td>
<td>Wako Pure Chemical Industries</td>
<td>169-04245</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Na2HPO4&middot;12H2O</td>
<td>Wako Pure Chemical Industries</td>
<td>500-04195</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MgCl2 &middot;6H2O</td>
<td>Wako Pure Chemical Industries</td>
<td>135-00165</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>CaCl2 &middot;2H2O</td>
<td>Wako Pure Chemical Industries</td>
<td>031-00435</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Penicillin G</td>
<td>Sigma-Aldrich</td>
<td>P-4687</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sodium pyruvate </td>
<td>Sigma-Aldrich</td>
<td>P-8574</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sucrose</td>
<td>Wako Pure Chemical Industries</td>
<td>196-00015</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>M16 medium</td>
<td>Sigma-Aldrich</td>
<td>M7292</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>M2 medium</td>
<td>Sigma-Aldrich</td>
<td>M7167</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Cryotube </td>
<td>Sumitomo Bakelite</td>
<td>MS-4501</td>
<td>&ldquo;Cryogenic Vial&rdquo;</td>
</tr>
</table>

cryopreservation of mouse embryos by ethylene glycol-based vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

Watch this Scientific Journal Video about Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification at JoVE.com

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

cryopreservation-mouse-embryos-ethylene-glycol-based

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Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

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Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...