Name: laser-assisted cytoplasmic microinjection in livestock zygotes
Uploaded: 2016-10-05T14:00:00
Description: Watch this Scientific Journal Video about Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes at JoVE.com

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

1. Производство Микропипетки <ol><li> Инъекции микропипетка <ol><li> Поместите боросиликатного стеклянный капилляр (наружный диаметр (OD): 1,0 мм, внутренний диаметр (ID): 0,75 мм) в микропипетки съемник (в центре держателей правый и левый капиллярных) и зафиксировать его. </li><li> С помощью соответству...

<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>

Микроинъекция зигот является хорошо установленный способ введения растворов в эмбрионов млекопитающих. С некоторыми вариациями в зависимости от вида и цели эксперимента, этот метод может быть использован в широком смысле. Мы покажем, как выполнить интрацитоплазматическую микроинъе?...

Цитоплазматическая микроинъекции 1-клеточных эмбрионов является очень мощной техникой. Он может быть использован для доставки любого раствора в эмбрион, чтобы, например, производят генных нокаутов для изучения функции генов или для создания генных отредактированный животных. Большинство сельскохозяйственно-зиготы соответствующих сельскохозяйственных животных имеют состав , очень высокую жирных кислот , что делает их цитоплазме непрозрачными и темные 1. Кроме того, они имеют достаточно эластичную мембрану плазмы (ПМ). Эти характеристики делают микроинъекции с использованием обычных пронуклеусов / цитоплазматической инъекции, используемый в сложных видов грызунов и часто неточны. Цитоплазматических микроинъекции имеет преимущества по сравнению с пронуклеарной микроинъекции так как легче выполнить , а также вызывает меньше повреждений инжектированных эмбрионов, что приводит к повышению жизнеспособности 2. Общей целью данного протокола является демонстрация успешного способа доставки решений в цитоплазму зиготы сельскохозяйственных животных. Для того, чтобы быть в состоянии выполнитьцитоплазматический микроинъекции с высокой эффективностью на эмбрионов скота, лазер используется для создания отверстия в пеллюцида (ZP), а затем стакан игла с тупым концом используется для микроинъекции. Эта стратегия направлена ​​на снижение механических повреждений отпечатаны на эмбрион во время инъекции. Затем, стремление цитоплазматического содержимого внутри инъекционной иглы позволяет эффективно и уверенно поломку ПМ, удерживающее раствор подается в цитоплазму зародыша. Эта методика уже успешно используется в эмбрионы крупного рогатого скота для доставки миРНК в зиготического цитоплазме 3,4 и генерировать мутации , используя кластерные регулярно interspaced короткие палиндромных повторы (CRISPR) CRISPR ассоциированной системы / 9 Система 5 (cas9). Он также подходит (с незначительными изменениями) , чтобы придать бычьи кучевые огороженный ооциты 6. Здесь мы описываем наш протокол инъекции поставляя краситель, который может быть применим к любой инъекции DESIRED раствор в зиготу, и показать, что с помощью этой техники вызывает минимальное лизис и не влияет на раннее развитие эмбриона.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micropipette puller</td>
			<td style="width:143px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Glass capillary</td>
			<td style="width:143px;">Sutter instruments</td>
			<td style="width:119px;">B100-75-10</td>
			<td style="width:257px;">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 height="20">
			<td height="20" style="height:20px;width:268px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:119px;">MF-9</td>
			<td>Equipped with 10X magnification lense.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micromanipulator</td>
			<td style="width:143px;">Nikon/ Narishige</td>
			<td>NT88-V3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Inverted microscope</td>
			<td style="width:143px;">Nikon</td>
			<td>TE2000-U</td>
			<td style="width:257px;">Equipped with 4x, 20x lenses and with a laser system.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser</td>
			<td style="width:143px;">Research Instruments</td>
			<td>7-47-500</td>
			<td>Saturn 5 Active laser.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Microdispenser</td>
			<td>Drummond</td>
			<td>3-000-105</td>
			<td style="width:257px;">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 height="100">
			<td height="100" style="height:100px;width:268px;">60mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430166</td>
			<td style="width:257px;">Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micropipettes with the micromanipulator easier.&#160;</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:268px;">35mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430165</td>
			<td style="width:257px;">These dishes are used for culturing the embryos in 50&#956;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 hours prior to transfering the embryos to them.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Incubator</td>
			<td style="width:143px;">Sanyo</td>
			<td style="width:119px;">MCO-19AIC</td>
			<td style="width:257px;">Any incubator that can be set to 38.5&#176;C 5% CO2 conditions can be used.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Stereomicroscope</td>
			<td style="width:143px;">Nikon</td>
			<td>SMZ800</td>
			<td style="width:257px;">Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10x magnification can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control Unit HT</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0400</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heated Microscope Stage</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0003</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Dextran-Red</td>
			<td>Thermo Scientific</td>
			<td>D1828</td>
			<td>A sterile 10mg/ml solution is used to inject.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:268px;">Mineral Oil</td>
			<td style="width:143px;">sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:257px;">Keep the mineral oil at room temperature and&#160; protected from light using foil paper.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">KSOMaa Evolve Bovine</td>
			<td style="width:143px;">Zenit</td>
			<td style="width:119px;">ZEBV-100</td>
			<td style="width:257px;">Supplemented with 4mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hours before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">FBS</td>
			<td style="width:143px;">Gemini-Bio</td>
			<td style="width:119px;">100-525</td>
			<td>Use a stem-cell qualified FBS.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Zygotes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Zygotes are injected 17-20 hpf and can be in-vitro- or in-vivo-derived.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td style="width:257px;">Final concentration: 107.7mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5405</td>
			<td style="width:257px;">Final concentration: 7.16mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KH2PO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td style="width:257px;">Final concentration: 1.19mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MgCL2 6H2O</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2393</td>
			<td style="width:257px;">Final concentration: 0.49mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium DL-lactate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">L4263</td>
			<td style="width:257px;">Final concentration: 5.3mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">CaCl2-2H2O&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">C7902</td>
			<td style="width:257px;">Final concentration: 1.71mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">D-(&#8722;)-Fructose&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">F3510</td>
			<td style="width:257px;">Final concentration: 0.5mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">HEPES&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:257px;">Final concentration: 21mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MEM-NEAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BME-EAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">B6766</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaHCO3</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:257px;">Final concentration: 4mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium pyruvate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P4562</td>
			<td style="width:257px;">Final concentration: 0.33mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">35050</td>
			<td style="width:257px;">Final concentration: 1mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BSA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">A-3311</td>
			<td style="width:257px;">Final concentration: 1mg/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Gentamicin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">G-1397</td>
			<td style="width:257px;">Final concentration: 5&#956;g/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Water for embryo transfer</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">W1503</td>
			<td style="width:257px;">Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">SOF-HEPES medium</td>
			<td style="width:143px;">Made in the lab</td>
			<td style="width:119px;"></td>
			<td style="width:257px;">pH 7.3-7.4, 280&#177;10 mOs. Filter sterilized through a 22&#956;m filter can be stored in the fridge at 4&#176; C for 1 month. Warm in 37 &#176;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.

Лазерная помощь цитоплазматический микроинъекции является мощным и надежным протоколом для доставки решений в цитоплазму зиготы скота. На рисунке 3 показан общий контур зиготы до и после инъекции, а также общий контур техники. Декстран-красный используется ...

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.

Помощью лазера цитоплазматических микроинъекции в животноводстве зиготы

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

The overall goal of this procedure is to effectively deliver solutions into the cytoplasm of livestock zygotes using a blunt-end needle with the aid of a laser. This method is used in research and in the animal trans-genesis field, since one is associated with a genome-editing tool, such as a CRISPR Cas9 system, it can create new cows to study gene function and transgenic animals in a very simple and reliable way. The main advantage of this technique is that it can be used to deliver any solution into a cytoplasm of large animal's zygotes.With a handy person's injection efficiency minimalizes animal liver lomotil rates. First, prepare the injection micropipette by placing a borosilicate glass capillary into a micropipette puller and locking it into place. Use an appropriate program to pull the capillary so it results in a thin tip with a long taper.Then, carefully remove the pulled pipettes from the device and place them onto a microforge in a horizontal position. Bring the pulled pipette and the microforge heater filament, with it's 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 of five micrometers.Adjust the heat to about 45%and gently bring the pipette down so it touches the filament. Then, briefly activate the heater. 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.Setting the right temperature is key in this step. Temperatures too high will bend the pipette, while temperatures too low will not be sufficient for cutting the pipette. Next, make an approximate 30 degree angle near the pipette tip by positioning the tip about 10 micrometers away from the heater filament.Set the temperature to 60%and activate the heater so that the pipette bends over the filament to the desired angle. Using the settings shown here, pull a glass capillary that results in a micro-pipette having a thin tip with a long, even taper and parallel walls. Carefully remove the pulled pipette from the puller device and place it on the microforge holder in a horizontal position.Use the eyepiece reticle to measure the pipette diameter and move the pipette until its diameter over the filament reaches 180 micrometers. Mark the glass capillary with a diamond-tipped pen and then gently press on the pulled tip to break the glass. This should result in a straight cut.Next, move the pipette to a vertical position, with its tip close to the filament. Set the temperature of the microforge to 60%and fire-polish the tip of the pipette, using standard techniques until the inner diameter reaches the target size. Measure the inner diameter of the pipette, which should be 40 micrometers.Using the same procedure as before, bend the tip to a 30 degree angle. Insert the holding pipette into the left micro-manipulator holder and the injection pipette into the right micro-manipulation holder. Then, fill the injection pipette with oil.Use the micro-manipulator controls to bring the pipettes into the center of the microscope's field of view. Using 4X magnification, check that the micro-pipette tips are at the correct angle so that they are parallel to the bottom of the dish. Place a 50 microliter drop of 37 degree celsius SOF-HEPES solution, that is supplemented with 20%FBS onto the center of the lid of a 100 millimeter petri dish.Then, place a one to two microliter drop of the solution to be injected close to the injection drop. Next, cover the drops and the injection plate using approximately 10 milliliters of mineral oil. Place the plate on the microscope stage and use a microdispenser to load about 20 to 30 zygotes in the upper side of the injection drop.Using the 4X objective, load the solution to be injected into the injection pipette by applying negative pressure to the microinjector. Load a sufficient amount of solution to inject two to three zygotes and then move to the injection drop. Within the injection drop, move the holding pipette so the tip gets close to a zygote.Apply negative pressure with the holding micro-injector so the zygote gets fixed to the holding pipette. Once the zygote is secure, change to a 20X objective. Check that the injection pipette is positioned on the embryo'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. Next, use software to place the laser vertical in the zona pellucida where the hole will be made. Once correctly positioned, use the laser control window and click on the FIRE button.Ensure that the size of the hole is large enough to create an opening in the zona pellucida for the needle to pass through without damaging the plasma membrane. Pass the injection needle through the hole in the zona pellucida and make contact with the plasma membrane. Continue pushing the pipette forward until the needle is 3/4 of the way into the embryo.Observe the position of the meniscus at the solution oil interphase. This will be used to consistently control the injection volume. Apply negative pressure on the injection pipette to aspirate the plasma membrane and cytoplasm into the injection needle.Continue until the movement of cytoplasm in the needle accelerates or when cytoplasm content, mixing with the injection solution, can be seen. These will indicate breakage of the plasma membrane. Inject back the cytoplasmic content, followed by the solution into the cytoplasm of the zygote, by applying positive pressure until the meniscus at the solution oil interphase has advanced one zygote's diameter equivalent past the starting point.Then, change to a 4X objective, and move the injected embryo to the lower side of the injection drop. Release the embryo by applying positive pressure on the holding micro-injector. Collect the injected embryos using a micro-dispenser.Wash the injected embryos by moving them through the three drops of SOF-HEPES and culture them, as described in the accompanying text protocol. Successful delivery of the solution is visible in the fluorescent image, where dexion red is used to track the site of injection and injection efficiency and accuracy. Here, the dye is homogeneously distributed in the cytoplasm, which is typical when using this technique.This protocol has minimal lysis rates in bovine and ovine zygotes. Few of the injected embryos were lysed in cattle, or sheep as a result of microinjection. Also, there are no statistically significant differences in blastocyst development rates in the control versus injected groups for both bovine and ovine embryos.Once mastered, this technique can be done at the rate of two embryos per minute. With a handy person injection efficiency, minimalizes animal blastocyst rates. After watching this video, you should understand how to perform laser-assisted intracytoplasmic micro-injection in livestock zygotes.And also how to use positive and negative suction pressure to assure the rupture of the plasma membrane for an efficient delivery.

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

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

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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

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Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

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Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

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Определения скорости оплодотворение яйца Bemisia tabaci с помощью цитогенетического техника

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Исследование транскрипционной роли интронического глушителя RUNX1 от CRISPR/Cas9 Рибонуклеопротеин в острых миелоидных клеток лейкемии

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

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Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...