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 ...

Research

JoVE Journal

Genetics

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

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

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

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

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

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