Work related to this technique is supported by NIH/NICHD RO1 HD070044 and USDA/NIFA Hatch projects W-3171 and W-2112.

1. Micropipette Production
<ol>
	<li>Injection micropipette
	<ol>
		<li>Place a borosilicate glass capillary (outer diameter (OD): 1.0 mm, inner diameter (ID): 0.75 mm) in a micropipette puller (in the center of the right and left capillary holders) and lock it.</li>
		<li>Use an appropriate program to pull the glass capillary so it results in a thin tip with a long tapper. (Example: Heat: 825; Pull: 30; Velocity: 120; Time: 200; Pressure: 500).</li>
		<li>Carefully remove the pulled pipettes from the device and place it on a microforge in a horizontal position.</li>
		<li>Bring the pulled pipette and the microforge heater filament with its glass bead into focus. Use the eyepiece reticle to measure the desired thickness and move the pipette horizontally until the heater filament reaches the target inner diameter (5 &#181;m).</li>
		<li>Adjust the heat to about 45% and gently bring the pipette down so it touches the filament. Activate the heater briefly. This will slightly melt the pipette so it adheres to the filament and upon cooling, the pipette will break at the contact point generating a straight cut. 
		Note: Setting the appropriate temperature is key in this step since temperatures too high will make the pipette bend and thus a straight cut will not be possible and temperatures too low will not be sufficient to melt the pipette (Figure 1A).</li>
		<li>Make an approximate 30&#176; angle near the pipette tip by positioning it about 10 &#181;m away from the heater filament. Set the temperature to 60% and activate the heater. 
		Note: This will bend the pipette over the filament. This angle is desired so the tip of the needle is parallel to the surface of the injection plate when mounted into the injector (Figure 1B).</li>
		<li>Handle microinjection pipettes with caution as they are extremely sharp and fragile. </li>
	</ol>
	</li>
	<li>Holding micropipette
	<ol>
		<li>Place a borosilicate glass capillary (OD: 1.0 mm, ID: 0.75 mm) in a micropipette puller (in the center of the right and left capillary holders) and lock it.</li>
		<li>Use an appropriate program to pull the glass capillary so it results in a thin tip with a long even taper and parallel walls (Example: Heat: 815; Pull: 20; Velocity: 140; Time: 175; Pressure: 200).</li>
		<li>Carefully remove the pulled pipette from the puller device and place it on the microforge holder in a horizontal position. Adjust the focus on the heater filament and pipette.</li>
		<li>Use the eyepiece reticle to measure the pipette diameter and move the pipette until its diameter over the filament reaches 180 &#181;m.</li>
		<li>At the indicated size, mark the glass capillary with a diamond tip pen, and then gently press on the pulled tip to break the glass. This should result in a straight cut (Figure 1C).</li>
		<li>Move the pipette to a vertical position with its tip close to the filament. Set the temperature of the microforge to 60%.</li>
		<li>Fire-polish the tip of the pipette using standard techniques7 until it reaches an ID of 40 &#181;m. Use the eyepiece reticle to check the ID (Figure 1D).</li>
		<li>Make an angle on the pipette.
		<ol>
			<li>Bring the pipette back to a horizontal position about 10 &#181;m away from the filament and 5 mm away from its tip. Set the temperature to approximately 60% and activate the heater to bend the pipette over the filament. Continue heating until a desired angle (of about 30o) is reached. Check the angle visually (Figure 1E). 
			Note: The pipette is usually mounted to the microinjector at an approximate angle of 60o with respect to the bottom of the injection dish. The tip of the pipette is bent so it is parallel to the bottom of the dish, which is necessary for the correct injection of the embryo at its middle plane.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Micromanipulator Setup
<ol>
	<li>Check that the microinjectors are fully loaded with oil and that there are no air bubbles in the system (bubbles in the microinjector prevent fine control of injection).</li>
	<li>Check that the micromanipulators are in the center position. This will allow for a wide range of movement of the pipettes.</li>
	<li>Insert the holding pipette into the left micromanipulator holder and the injection pipette into the right micromanipulation holder.</li>
	<li>Allow oil to enter into the pipettes by capillarity and check that the system is working properly by moving oil up and down inside the pipette using the microinjector controls. 
	Note: If there is no movement of oil inside the needles, there might be a clog. In this case, use a new needle.</li>
	<li>Use the micromanipulator controls to bring the pipettes into the center of the microscope&#39;s field of view. Using 4X magnification, check that the micropipette tips are in the correct angle (parallel to the bottom of the dish). 
	Note: A correct setup of the needles is key for a successful injection and for results consistency.</li>
	<li>Calibrate the laser system following the manufacturer&#39;s calibration manual.</li>
</ol>
3. Preparation of Injection Dish (Figure 2)
<ol>
	<li>Place a 50 &#181;l drop of warmed (37 &#176;C) SOF-HEPES (composition detailed in the materials section) supplemented with 20% fetal bovine serum (FBS) on the center of the lid of a 100 mm petri dish. Place a 1 - 2 &#181;l drop of the solution to be injected close to the injection drop. Ensure that the embryos do not cool down below RT. 
	Note: In this protocol we will add the Dextran-Red dye to the injecting solution to be able to visualize the site of injection and track it later.</li>
	<li>Cover the drops in the injection plate using approximately 10 ml of mineral oil.</li>
	<li>Place the injection dish on the stage of the inverted microscope and bring the pipettes into the injection drop. Check that the needles are in focus inside the drop. Adjust their height as needed using the micromanipulator controls.</li>
	<li>Using a microdispenser, load about 20 - 30 zygotes (17 - 20 hr post-fertilization (hpf)) in the upper side of the injection drop. Perform injection at room temperature so the number of embryos in the injection drop is determined by the speed at which the person can inject them. Do not load more embryos than the ones that can be injected in 30 min.
	<ol>
		<li>To load the embryos into the microdispenser, depress the plunger fully and immerse the tip into the solution containing the embryos. Touch the embryos to be picked up with the tip of the glass capillary (one at the time) and slowly release the plunger so each embryo enters into the capillary. Repeat this until all the embryos are picked up or the microdispenser capacity is full.</li>
		<li>To release the loaded embryos, depress the plunger gently until the entire volume containing the embryos is released from the capillary.</li>
	</ol>
	</li>
</ol>
4. Microinjection
<ol>
	<li>Using the 4X objective, load the solution to be injected into the injection pipette by applying negative pressure (aspirating). Load sufficient solution to inject 2 - 3 zygotes. Then move to the injection drop.</li>
	<li>Within the injection drop, move the holding pipette so the tip gets close to a zygote. Apply negative pressure (aspirate) with the holding microinjector so the zygote gets fixed to the holding pipette and change to a 20X objective.</li>
	<li>Once the zygote is attached to the holding pipette, check its quality using the injecting pipette to gently move the embryo around without detaching it. A good quality zygote should have the two polar bodies (although sometimes only one is seen) and a homogeneous cytoplasm. Do not inject abnormal zygotes (Figure 3A).</li>
	<li>If necessary, reposition the embryo for a proper injection location. A good positioning would be that in which there is some space between the ZP and the PM, so the PM does not get damaged by the laser.</li>
	<li>Check that the injection pipette is positioned on the embryo&#39;s mid-plane by gently touching the zygote on the site of injection. If it is not on its mid-plane, the needle will tend to make the embryo rotate instead of puncture. Adjust the height of the injection pipette as needed.</li>
	<li>Make a hole in the ZP using a laser (Figure 3B).
	<ol>
		<li>Using the laser&#39;s software place the laser reticle in the ZP where the hole will be made (on the opposite side to where the embryo is attached to the holding pipette). Using the laser control window click on the fire button. Ensure that the size of the hole is large enough to create an opening in the ZP for the needle to pass through without damaging the PM (Example: 0.662 msec Pulse width/ 6.9 &#181;m Hole size).</li>
	</ol>
	</li>
	<li>Pass the injection needle through the hole in the ZP and make contact with the PM. Continue pushing the pipette forward until the needle is &#190; of the way into the embryo (Figure 3C).</li>
	<li>Observe the position of the meniscus at the solution-oil interphase, as this will be used to consistently control injection volume.</li>
	<li>Apply negative pressure (aspirate) on the injection pipette, which will result in PM and cytoplasm being aspirated into the injection needle. Continue until the movement of cytoplasm into the needle accelerates or when cytoplasm content mixing up with injection solution can be seen. These will indicate breakage of the PM (Figure 3D).</li>
	<li>Inject back the cytoplasmic content followed by the solution into the cytoplasm of the zygote by applying positive pressure (injecting), until the meniscus at the solution-oil interphase has advanced one zygote&#39;s diameter equivalent past the starting point. 
	Note: Under our conditions, this represents approximately 7 pl of injected solution (Figure 3E).</li>
	<li>Change to a 4X objective and move the injected embryo/s to the lower side of the injection drop. Release the embryo by applying positive pressure on the holding microinjector. Keep the non-injected embryos on the upper side and the injected ones on the lower side of the drop to help keep track of the injected/non-injected embryos and work efficiently.</li>
</ol>
5. Embryo Recovery and Injection Results
<ol>
	<li>Make 3 drops of approximately 200 &#181;l in a new dish with pre-warmed SOF-HEPES. Collect the injected embryos using a microdispenser (as described above). Wash the injected embryos by moving them through the 3 SOF-HEPES drops.</li>
	<li>Count the embryos lysed during microinjection and discard them. 
	Note: A lysed zygote can be easily distinguished from an intact one since the first one appears translucent, fill out the whole ZP, and it usually has cytoplasmic content leaking to the outside of the embryo.</li>
	<li>Optionally, check the microinjection efficiency using a fluorescent microscope. Be aware that UV light exposure can induce embryo damage that can affect subsequent development.</li>
	<li>Culture groups of 25 injected embryos in 50 &#181;l drops of potassium simplex optimization medium (KSOM) supplemented with 4 mg/ml bovine serum albumin (BSA) under mineral oil at 38.5 &#176;C, 5% CO2 in air, and humidity to saturation.</li>
	<li>Supplement the culture media with 5% FBS two days after injection.</li>
	<li>Check blastocyst (BL) rates 7 days after injection. Calculate BL rates as total number of blastocyst embryos/total number of embryos cultured in that drop.</li>
</ol>

<ol>
	<li>McEvoy, T., Coull, G., Broadbent, P., Hutchinson, J., Speake, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+of+lipids+in+immature+cattle,+pig+and+sheep+oocytes+with+intact+zona+pellucida.">Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.</a> J Reprod Fertil. 118 (1), 163-170 (2000).</li><li>Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+efficiency+of+introducing+foreign+DNA+into+mice+by+microinjecting+eggs.">Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.</a> Proc Natl Acad Sci U S A. 82 (13), 4438-4442 (1985).</li><li>Ross, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parthenogenetic+activation+of+bovine+oocytes+using+bovine+and+murine+phospholipase+C+zeta.">Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta.</a> BMC Dev Biol. 8 (1), (2008).</li><li>Canovas, J., Cibelli, J. B., Ross, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jumonji+domain-containing+protein+3+regulates+histone+3+lysine+27+methylation+during+bovine+preimplantation+development.">Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development.</a> Proc Natl Acad Sci U S A. 109 (7), 2400-2405 (2012).</li><li>Bogliotti, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4+Developmental+outcomes+and+effiency+of+two+CRISPR/Cas9+microinjection+methods+in+bovine+zygotes.">4 Developmental outcomes and effiency of two CRISPR/Cas9 microinjection methods in bovine zygotes.</a> Reprod Fertil Dev. 27 (1), 94-94 (2015).</li><li>Bakhtari, A., Ross, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DPPA3+prevents+cytosine+hydroxymethylation+of+the+maternal+pronucleus+and+is+required+for+normal+development+in+bovine+embryos.">DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos.</a> Epigenetics. 9 (9), 1271-1279 (2014).</li><li>Yaul, M., Bhatti, R., Lawrence, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+process+of+polishing+borosilicate+glass+capillaries+used+for+fabrication+of+in-vitro+fertilization+(iVF)+micro-pipettes.">Evaluating the process of polishing borosilicate glass capillaries used for fabrication of in-vitro fertilization (iVF) micro-pipettes.</a> Biomed Microdevices. 10 (1), 123-128 (2008).</li><li>Sutter Instrument. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pipette Cookbook 2015 P-97 &amp; P-1000 Micropipette Pullers. , (2015).</li><li>Sutter Instrument. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-97 Flaming/BrownTM Micropipette Puller Operation Manual Rev. 2.30 - DOM (20140825). , (2014).</li><li>Cibelli, J. B., Lanza, R. P., Campbell, K. H. S., West, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of cloning. , (2002).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+reporter+gene+after+microinjection+of+mammalian+artificial+chromosomes+into+pronuclei+of+bovine+zygotes.">Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.</a> Mol Reprod Dev. 60 (4), 433-438 (2001).</li></ol>

The authors have nothing to disclose.

Microinjection of zygotes is a well-established method for introducing solutions into mammalian embryos. With some variations dependent on the species and the aim of the experiment, this technique can be broadly used. We show how to perform intracytoplasmic microinjection using a laser to assist the entrance of a blunt-end micropipette. Zygotes of some livestock species (such as cattle, sheep, and pig) have a dark cytoplasm, hindering the visualization of the injection pipette once inside the embryo. Also, their plasma m...

Cytoplasmic microinjection of 1-cell embryos is a very powerful technique. It can be used for delivering any solution into the embryo to, for example, produce gene knock-outs to study gene function or to generate gene-edited animals. Most agriculturally-relevant farm animal zygotes have a very high fatty acid composition that makes their cytoplasm opaque and dark1. They also have a fairly elastic plasma membrane (PM). These characteristics make microinjection using conventional pronuclear/cytoplasmic injection as used in rodent species challenging and often inaccurate.
Cytoplasmic microinjection has advantages over pronuclear microinjection since it is easier to perform and also causes less damage to the injected embryos, resulting in higher viability2. The overall goal of this protocol is to demonstrate a successful method for delivering solutions into the cytoplasm of farm animal zygotes. To be able to perform cytoplasmic microinjection with high efficiency on livestock embryos, a laser is used to generate a hole in the zona pellucida (ZP) and then a blunt-end glass needle is used for the microinjection. This strategy aims to reduce the mechanical damage imprinted on the embryo during injection. Then, aspiration of cytoplasmic content inside the injection needle allows efficient and confident breakage of the PM ensuring that the solution is delivered into the cytoplasm of the embryo.
This technique has already been successfully used in bovine embryos to deliver siRNA into the zygotic cytoplasm3,4 and to generate mutations using the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated system 9 (Cas9) system5. It is also suitable (with minor modifications) to inject bovine cumulus-enclosed oocytes6. Here, we describe our injection protocol delivering a dye, that can be applicable to injecting any desired solution into the zygote, and show that using this technique causes minimal lysis and does not affect early embryo development.

<table><tbody><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Glass capillary</td><td>Sutter instruments</td><td>B100-75-10</td><td>These capillaries are used for making the holding and injecting pipettes. Any thick/standard wall borosilicate tubing without filament can be used.</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-9</td><td>Equipped with 10X magnification lense.</td></tr><tr><td>Micromanipulator</td><td>Nikon/ Narishige</td><td>NT88-V3</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>TE2000-U</td><td>Equipped with 4X, 20X lenses and with a laser system.</td></tr><tr><td>Laser</td><td>Research Instruments</td><td>7-47-500</td><td>Saturn 5 Active laser.</td></tr><tr><td>Microdispenser</td><td>Drummond</td><td>3-000-105</td><td>The microdispenser is used to move the embryos. A p10 pipette can also be used but loading as minimal volume as possible.</td></tr><tr><td>100 x 15 mm culture dish</td><td>Falcon</td><td>351029</td><td>Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micromanipulator easier. The lid of a 60 mm culture dish can also be used.</td></tr><tr><td>35 mm culture dish</td><td>Corning</td><td>430165</td><td>These dishes are used for culturing the embryos in 50 &amp;mu;l drops covered with mineral oil. Alternatively, a 4 well dish can also be used. Regardless of the dish chosen to culture the embryos, they always have to be equilibrated in the incubator for at least 4 hr prior to transfering the embryos to them.</td></tr><tr><td>Incubator</td><td>Sanyo</td><td>MCO-19AIC</td><td>Any incubator that can be set to 38.5 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; conditions can be used.</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ800</td><td>Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10X magnification can be used.</td></tr><tr><td>Control Unit HT</td><td>Minitube</td><td>12055/0400</td><td>Heating system attached to the stereomicroscope.</td></tr><tr><td>Heated Microscope Stage</td><td>Minitube</td><td>12055/0003</td><td>Heating system attached to the stereomicroscope.</td></tr><tr><td>Dextran-Red</td><td>Thermo Scientific</td><td>D1828</td><td>A sterile 10 mg/ml solution is used to inject.</td></tr><tr><td>Mineral Oil</td><td>sigma</td><td>M8410</td><td>Keep the mineral oil at room temperature and&amp;nbsp; protected from light using foil paper.</td></tr><tr><td>KSOMaa Evolve Bovine</td><td>Zenit</td><td>ZEBV-100</td><td>Supplemented with 4 mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hr before use.</td></tr><tr><td>FBS</td><td>Gemini-Bio</td><td>100-525</td><td>Use a stem-cell qualified FBS.</td></tr><tr><td>Zygotes</td><td></td><td></td><td>Zygotes are injected 17 - 20 hpf and can be &lt;em&gt;in-vitro&lt;/em&gt;- or &lt;em&gt;in-vivo&lt;/em&gt;-derived.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td>Final concentration: 107.7 mM. Component of SOF-HEPES medium.</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td>Final concentration: 7.16 mM. Component of SOF-HEPES medium.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td>Final concentration: 1.19 mM. Component of SOF-HEPES medium.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2393</td><td>Final concentration: 0.49 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Sodium DL-lactate</td><td>Sigma</td><td>L4263</td><td>Final concentration: 5.3 mM. Component of SOF-HEPES medium.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>C7902</td><td>Final concentration: 1.71 mM. Component of SOF-HEPES medium.</td></tr><tr><td>D-(&amp;minus;)-Fructose&amp;nbsp;</td><td>Sigma</td><td>F3510</td><td>Final concentration: 0.5 mM. Component of SOF-HEPES medium.</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td>Final concentration: 21 mM. Component of SOF-HEPES medium.</td></tr><tr><td>MEM-NEAA</td><td>Sigma</td><td>M7145</td><td>Final concentration: 1x. Component of SOF-HEPES medium.</td></tr><tr><td>BME-EAA</td><td>Sigma</td><td>B6766</td><td>Final concentration: 1x. Component of SOF-HEPES medium.</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>Final concentration: 4 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P4562</td><td>Final concentration: 0.33 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050</td><td>Final concentration: 1 mM. Component of SOF-HEPES medium.</td></tr><tr><td>BSA</td><td>Sigma</td><td>A-3311</td><td>Final concentration: 1 mg/ml. Component of SOF-HEPES medium.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G-1397</td><td>Final concentration: 5 &amp;mu;g/ml. Component of SOF-HEPES medium.</td></tr><tr><td>Water for embryo transfer</td><td>Sigma</td><td>W1503</td><td>Component of SOF-HEPES medium.</td></tr><tr><td>SOF-HEPES medium</td><td>Made in the lab</td><td></td><td>pH 7.3 - 7.4, 280 &amp;plusmn; 10 mOs. Filter sterilized through a 22 &amp;mu;m filter can be stored in the fridge at 4 &amp;deg;C for 1 month. Warm in 37 &amp;deg;C water bath before use.</td></tr></tbody></table>

laser-assisted cytoplasmic microinjection in livestock zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

Laser-assisted cytoplasmic microinjection is a powerful and reliable protocol to deliver solutions into the cytoplasm of livestock zygotes. Figure 3 shows a general outline of the zygotes before and after injection as well as the overall outline of the technique. Dextran-red is used as injecting solution to allow tracking site of injection and injection efficiency and accuracy. Successful delivery of the solution is illustrated in Figure 4 showing a recen...

Watch this Scientific Journal Video about Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes at JoVE.com

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

laser-assisted-cytoplasmic-microinjection-in-livestock-zygotes

Research

JoVE Journal

Genetics

9.0K Views.  University of California, Davis. This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Video: Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Developmental Biology

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biology

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

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Basic Biology

Biology II: Mouse, Zebrafish, and Chick

Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery

Source: Martin, N. P., et al. <a href="https://www.jove.com/v/58327">Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs.</a> J. Vis. Exp. (2018).
In this video, we demonstrate the laser-assisted permeation of the protective layer of zona pellucida in mouse fertilized eggs for facilitating lentiviral gene delivery. Lentivirus enables the generation of transgenic animals with a gene of interest stably integrated into their genome.

Source: Martin, N. P., et al. Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs. J. Vis. Exp. (2018).
In this video, we demonstrate the ...

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

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