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. Micropipette उत्पादन <ol><li> इंजेक्शन micropipette <ol><li> एक borosilicate ग्लास केशिका (1.0 मिमी, भीतरी व्यास (आईडी): बाहरी व्यास (ओवर ड्राफ्ट) 0.75 मिमी) की जगह एक micropipette खींचने में (दाएं और बाएं केशिका धारकों के केंद्र में है) और यह ताला। </li><li> ...

<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>

युग्मनजों के microinjection स्तनधारी भ्रूण में समाधान शुरू करने के लिए एक अच्छी तरह से स्थापित विधि है। कुछ बदलाव प्रजातियों और प्रयोग के उद्देश्य पर निर्भर है के साथ, इस तकनीक को मोटे तौर पर इस्तेमाल किया जा सक...

1-सेल भ्रूण के cytoplasmic microinjection एक बहुत शक्तिशाली तकनीक है। यह भ्रूण में किसी भी समाधान प्रदान करने के लिए, उदाहरण के लिए, जीन समारोह का अध्ययन करने के लिए या जीन से संपादित जानवरों उत्पन्न करने के लिए उत्पादन जीन दस्तक बहिष्कार के लिए इस्तेमाल किया जा सकता है। अधिकांश कृषि प्रासंगिक खेत पशु युग्मनजों एक बहुत ही उच्च फैटी एसिड संरचना है कि उनके कोशिका द्रव्य अपारदर्शी और अंधेरे 1 बनाता है। उन्होंने यह भी एक काफी लोचदार प्लाज्मा झिल्ली (प्रधानमंत्री) है। इन विशेषताओं पारंपरिक pronuclear / cytoplasmic इंजेक्शन का उपयोग कर के रूप में कृंतक प्रजातियों चुनौतीपूर्ण और अक्सर गलत में इस्तेमाल microinjection बनाते हैं। क्योंकि यह प्रदर्शन करने के लिए आसान है और यह भी, इंजेक्शन भ्रूण को कम नुकसान का कारण बनता है उच्च व्यवहार्यता 2 में जिसके परिणामस्वरूप cytoplasmic microinjection pronuclear microinjection से अधिक लाभ है। इस प्रोटोकॉल के समग्र लक्ष्य के खेत पशु युग्मनजों की कोशिका द्रव्य में समाधान देने के लिए एक सफल विधि का प्रदर्शन करने के लिए है। प्रदर्शन करने में सक्षम होना करने के लिएपशुधन भ्रूण पर उच्च दक्षता के साथ cytoplasmic microinjection, एक लेजर zona pellucida (जिला परिषद) और फिर एक कुंद अंत गिलास सुई microinjection के लिए प्रयोग किया जाता है में एक छेद उत्पन्न करने के लिए प्रयोग किया जाता है। इस रणनीति के यांत्रिक इंजेक्शन के दौरान भ्रूण पर अंकित नुकसान को कम करना है। फिर, इंजेक्शन सुई अंदर cytoplasmic सामग्री की आकांक्षा सुनिश्चित करना है कि समाधान भ्रूण की कोशिका द्रव्य में कर दिया है प्रधानमंत्री के कुशल और विश्वास टूटना अनुमति देता है। इस तकनीक को पहले से ही सफलतापूर्वक युग्मज कोशिका द्रव्य 3,4 में siRNA देने के लिए और क्लस्टर नियमित रूप से interspaced कम मुरजबंध संबंधी दोहराता (CRISPR) / CRISPR जुड़े प्रणाली 9 (Cas9) प्रणाली का उपयोग करते हुए 5 म्यूटेशन उत्पन्न करने के लिए गोजातीय भ्रूण में इस्तेमाल किया गया है। यह भी उपयुक्त (मामूली संशोधनों के साथ) गोजातीय मेघपुंज संलग्न oocytes 6 इंजेक्षन है। यहाँ, हम हमारे इंजेक्शन एक डाई पहुंचाने प्रोटोकॉल का वर्णन है, कि किसी भी des इंजेक्शन लगाने के लिए लागू हो सकता हैयुग्मनज में iRed समाधान, और पता चलता है कि इस तकनीक का उपयोग कम से कम सेल का कारण बनता है और जल्दी भ्रूण के विकास को प्रभावित नहीं करता।

<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.

लेजर की मदद से cytoplasmic microinjection पशुधन युग्मनजों की कोशिका द्रव्य में समाधान देने के लिए एक शक्तिशाली और विश्वसनीय प्रोटोकॉल है। चित्रा 3 से पहले और इंजेक्शन के साथ ही तकनीक के समग्र रूपरेख?...

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.

लेजर की मदद से पशुधन युग्मनज में cytoplasmic microinjection

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

पर हौसले अलग मानव विलस Cytotrophoblasts siRNA अभिकर्मक और EMSA विश्लेषण

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

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

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

दुनिया भर में कृषि कीट कीट में भ्रूण Microinjection के माध्यम से न्यूक्लिक एसिड की डिलिवरी,<em&gt; Ceratitis capitata</em

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic यादृच्छिक mutagenesis में Histone-miniSOG का प्रयोग<em&gt; सी। एलिगेंस</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

पालन ​​और मिटायें बीटल में डबल असहाय शाही सेना की मध्यस्थता जीन पछाड़ना,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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

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

Microinjection CRISPR/Cas9 प्रोटीन की चैनल कैटफ़िश, Ictalurus punctatus, जीन संपादन के लिए भ्रूण

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

भ्रूण Microinjection और प्रत्यारोपण तकनीक Nasonia vitripennis जीनोम हेरफेर के लिए

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

विशेषता हिस्one पोस्ट-translational में संशोधन परिवर्तन खमीर न्यूरोडेजेनेरेटिव प्रोटिनोपैथी मॉडल

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

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

एक कोशिका आनुवंशिक तकनीक का उपयोग कर बेमिसिया टैबची के अंडे निषेचन दर का निर्धारण

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

तीव्र माइलॉयड ल्यूकेमिया कोशिकाओं में CRISPR/Cas9 Ribonucleoप्रोटीन द्वारा एक RUNX1 Intronic साइलेंसर की ट्रांसक्रिप्शनल भूमिका की जांच

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

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

क्यूलेक्स क्विंक्विफासिटस में कुशल साइट-विशिष्ट म्यूटेनेसिस के लिए भ्रूण माइक्रोइंजेक्शन तकनीक

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