Name: trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates
Uploaded: 2018-05-29T13:00:00
Description: Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

This research was supported [in part] by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. Special thanks to Shyamal Peddada for statistical analysis. We also wish to thank Humphrey Yao, Gary Bird, and Yuki Arao for review of this manuscript, and Franco DeMayo for his continued support and advice.

All operations were done as part of the normal operation of the NIEHS Knock Out Mouse Core facility. All mice were kept on a 12:12 h light:dark cycle, and were feed a diet of NIH-31 and water ad libitum. All experiments were performed in accordance with the Animal Care and Use Committee of the National Institute of Environmental Health Sciences.
1. Animal Preparation
<ol>
	<li>Breed donor mice, B6(Cg)-Tyr&#60;c-2J&#62;/J female mice at 8 - 10 weeks of age naturally to B6(Cg)-Tyr&#60...

<ol>
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It has long been thought that any disturbance of the ICM could lead to mortality, in fact, current blastocyst microinjection protocols still warn of this12,13. What we have shown here is that microinjection through the ICM is not detrimental to the embryo, and increases the yield of chimeras.
The exact mechanism for this effect has not been identified. However, the mechanical disruption of the ICM, and the accompanying hypoblast, shoul...

For nearly 25 years, the creation of genetically targeted mice has been a slow process, often hampered by a bottleneck at the blastocysts ES cell microinjection stage for the generation of germline transmitting chimeras. Recent advancements, including CRISPR/Cas91,2 targeting in ES cells and high-throughput ES cell generation consortiums like KOMP, have improved the generation/availability of genetically modified ES cells. However, the generation of germline chimeras remains a bottleneck in creating genetically modified mice from these ES cells3,4. High-throughput ES cell generation projects have been plagued by high variability in ES cell quality, which is critical for successful creation of germline chimeras. Additionally, some of the commonly utilized ES cell lines are known for high aneuploidy, and difficult production of germline competent chimeras5.
Many methods have been developed for creating mice with either a higher chimera, or completely ES cell derived mice6,7,8,9,10. While each of these systems has its own merits, they also have their shortcomings. The generation of tetraploid chimeras, while generating 100% ES cell derived mice, is inefficient, and generally limited to outbred lines 5,6. Newer methods of injecting morula can yield high percentage chimeras, approaching 100%, but generally require significant additional equipment (lasers or piezos) and training10,11. In laboratories where these techniques are already in place, this new methodology may not be necessary.
This study&#39;s objective was to find a method that uses common techniques and readily available equipment to increase the rate of chimera generation, increasing the chance of germline transmission of the mutant allele to establish the subsequent mouse colony.

<table>
	<tbody>
		<tr>
			<td>ES Cell injection needle</td>
			<td>Humagen</td>
			<td>MSC-20-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NSET device</td>
			<td>Paratechs</td>
			<td>60010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (life technologies)</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (life technologies)</td>
			<td>10439-024</td>
			<td>lots should be individually tested for ES Cell compatibility.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulator</td>
			<td>Leica</td>
			<td>Micro manipulator</td>
			<td></td>
		</tr>
		<tr>
			<td>micro injector</td>
			<td>Eppendorf AG</td>
			<td>Cell Tram Air 5176</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>4 well dish</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm dish</td>
			<td>Sarstedt</td>
			<td>83.3901</td>
			<td></td>
		</tr>
		<tr>
			<td>B6(Cg)-Tyr&#60;c-2J&#62;/J</td>
			<td>Jackson Labs, Bar Harbor, Maine, USA</td>
			<td>000058</td>
			<td></td>
		</tr>
		<tr>
			<td>Swiss Webster mice</td>
			<td>Taconic, USA</td>
			<td>Tac:SW</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Dish</td>
			<td>MatTek Corp.</td>
			<td>P50G-0-30-F</td>
			<td></td>
		</tr>
	</tbody>
</table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Treating cell-line as the random effect, a mixed effects linear model was used to analyze the data in the software (e.g. SAS (version 9.2)). Each analysis controlled for the number of embryos transferred and the cell line. The second analysis also controlled for the number of pups that survived.
For this study, each ES Cell line and clone was injected both with traditional and TICM techniques, alternating between the two methods...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

The overall goal of this modified procedure is to increase chimeric production without incurring additional costs or long training periods. This method can help laboratories become more efficient in chimera production, especially in cases where the ES cells are less than optimal. The main advantage of this technique is that it requires no additional equipment and no specialized training.Demonstrating the procedure today will be Thomas Hagler. He's a biologist and graduate student who works in the core lab. To harvest the embryos, blot the uterus on a lab tissue to remove the excess blood and place the organ in a new 60 millimeter dish under a low magnification dissecting microscope.Using dissecting forceps to carefully and gently hold the uterus near the oviduct, insert a 25 gauge needle attached to a 10 milliliter syringe containing 10 milliliters of blast collection medium into the center of the uterus just beyond the tips of the forceps in as parallel a plane as possible. Apply pressure to the forceps to secure the needle within the uterus and gently flush approximately one milliliter of medium through the uterine horn. When all of the blastocysts have been expelled, assemble the mouth pipette apparatus and use the mouth pipette to transfer the individual embryos into single drops of blast collection medium in a second 60 millimeter dish.Then wash each embryo in a second drop of blast collection medium and transfer the embryos into a four-well dish until their injection. To begin, use a microinjector to apply vacuum to the injection needle. Place a dish of ES cells under the injection microscope where the injection needle and embryo holder pipette are attached.It is essential to the success of the injection to take the time to properly setup the microscope. This includes the proper amount of media in the needle and the correct alignment of the z-axis. Use a microinjector to apply vacuum to the injection needle.Keeping as little space between the cells as possible, draw in smoothly surfaced small to medium sized embryonic cells without obvious defects from the dish of embryos. After collecting at least 15 cells, position an embryo near the opening of the holder. Apply vacuum to secure the embryo to the holder and move the holding pipette up or down to adjust the z-axis until the embryo is just touching the slide.Fine focus on the zone of pellucida and adjust the z-axis of the injection needle until the embryonic cells near the tip of the needle are in focus. If needed, use the injection needle to gently roll the blastocyst until the inner cell mass is in the appropriate position for injection. Using a firm but controlled push, insert the injection needle through the zone of pellucida into the inner cell mass and apply a slight pressure to the microinjector of the injection needle to help keep the blastocoele from collapsing.Once the needle has penetrated the blastocoele, apply pressure to the microinjector to transfer all of the embryonic stem cells into embryo, pausing briefly as the bevel of the needle starts to exit the embryo to allow the pressure in the embryo to return to normal while still retaining the cells. When all of the blastocysts have been injected, use a nonsurgical embryo transfer device to deliver the embryos to embryonic stage 2.5 Swiss Webster mice according to the manufacturer's instructions and house the mice individually after each transfer through delivery and subsequent weaning. In this representative experiment, the pregnancy rates were unaltered between the trans inner cell mass injected and control traditionally injected groups and an equal number of pups survived to weaning from each group.Notably, while the number of mice at weaning remained unchanged, a significantly higher number of pups from the trans inner cell mass injected group displayed coat-color chimerism compared to the control injected group. The best part of this technique is that it only requires a simple change in the orientation of the embryo to facilitate greater production in chimeras. Proper setup of the microscope is essential.If the microscope and manipulators aren't setup properly and maintained well, the injection process will not work. If you're new to embryo injections, repetition is the only way to learn. For beginners, embryo handling tends to be the most difficult part of the procedure.Being able to move embryos efficiently from one dish to another without losing any is the most crucial part of the experiment. After watching this video, you should have a good understanding of how a simple change to your standard injection technique can help you become more efficient in chimera production.

Research

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