We thank Santiago Suarez for critical reading of the manuscript and Betty Cowgill for aid in photography. We also thank the anonymous reviewers for their critical feedback. This work is supported by the University of Delaware Research Fund.

1. Preparation of Protamine Sulfate (PS) Coated Dishes
<ol>
	<li>Prepare 1% solution of protamine sulfate (PS) by adding 0.5 g of PS to 50 ml of deionized, distilled water (ddH2O) in a 50 ml conical tube. Shake well at high speed on a bench shaker at room temperature for 1-2 hr to ensure complete dissolution of PS. This solution can be stored at 4 &#176;C for at least 3 months (make sure to completely dissolve gel-like precipitate before each use)3.</li>
	<li>Take a sleeve of 60 mm x 15 mm polysty...

<ol>
	<li>Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+nonviral+transfection+methods+for+foreign+gene+expression+in+early+chicken+embryos+in+ovo.">Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.</a> Biochem. Biophys.Res. Commun. 230, 376-380 (1997).</li><li>Peng, H., Wu, Y. Y., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Delivery+of+DNA+and+Morpholinos+into+Mouse+Preimplantation+Embryos+by+Electroporation.">Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation.</a> Plos One. 7, 13 (2012).</li><li>Ettensohn, C. A., Wray, G. A., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> eds. Development of sea urchins, ascidians, and other invertebrate deuterostomes: experimental approaches. 74, (2004).</li><li>Franks, R. R., Houghevans, B. R., Britten, R. J., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+introduction+of+cloned+DNA+into+the+sea+urchin+zygote+nucleus,+and+fate+of+injected+DNA.">Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA.</a> Development. 102, 287-299 (1988).</li><li>Kinsey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+signaling+pathways+in+zebrafish+development+by+microinjection.">Analysis of signaling pathways in zebrafish development by microinjection.</a> Methods Mol. Biol. 518, 67-76 (2009).</li><li>Kola, I., Sumarsono, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+in+vitro+transcribed+RNA+and+antisense+oligonucleotides+in+mouse+oocytes+and+early+embryos+to+study+the+gain-+and+loss-of-function+of+genes.">Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes.</a> Mol. 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A., Gasiorowski, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+gene+delivery.">Nonviral gene delivery.</a> Cold Spring Harb. Protoc. , (2011).</li><li>Hiramoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+division+without+mitotic+apparatus+in+sea+urchin+eggs.">Cell division without mitotic apparatus in sea urchin eggs.</a> Exp. Cell Res. 11, 630-636 (1956).</li><li>Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 25, (1982).</li><li>Gross, J. M., McClay, D. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offerings+from+an+Urchin.">Offerings from an Urchin.</a> Dev. Biol. 358, 285-294 (2011).</li><li>McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+sea+urchins.">Evolutionary crossroads in developmental biology: sea urchins.</a> Development. 138, 2639-2648 (2011).</li><li>Sodergren, E., 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=Research+article+-+The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">Research article - The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Roux, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B+synthesis+is+required+for+sea+urchin+oocyte+maturation.">Cyclin B synthesis is required for sea urchin oocyte maturation.</a> Dev. Biol. 256, 258-275 (2003).</li><li>Wong, J. L., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+modifications+at+fertilization:+regulation+of+dityrosine+crosslinking+by+transamidation.">Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation.</a> Development. 136, 1835-1847 (2009).</li><li>Shuster, C. B., Burgess, D. 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Microinjection is a powerful technique for delivering various treatments such as DNA, mRNA, recombinant proteins, loss of function and gain of function reagents, dyes and their combinations into eggs, embryos, and cells of various organisms1-7. However, several considerations should be kept in mind when designing a microinjection experiment.
It is critically important to consider the solubility of the delivered treatment and the injection volume. If the microinjected solution tends ...

Efficient and reproducible treatment delivery is one of the main methodological challenges for researchers. Several methods have been established to transiently deliver treatment solutions into the eggs, embryos, and cells. These methods include electroporation (based on a generating transient pores in the membrane using short electrical pulses)1,2, lipofection (delivery through the fusion of treatment-containing liposomes with the membrane)1, microparticle bombardment1 (DNA is precipitated on the micron-sized metal particles that are then used to penetrate the cells at high velocity), and transduction (virus is used as a delivery vehicle of transgenes). At the moment, microinjection is the only approach that holds the advantage of delivering any solution with 100% efficiency with minimal reagents. Moreover, a single injection solution can be composed of a complex cocktail of treatments. This technique has been used to successfully microinject the eggs and embryos from numerous species such as sea urchins3,4, zebra fish5, mouse6, frog7, and cattle8 as well as single cells in the tissue culture9. Single blastomeres injections at later developmental stages have also been conducted10-12.
The current methods of microinjections are based on the pressure-injection method that was initially described by Hiramoto10; however, great progress has been made towards optimization of this process. Excellent microinjection techniques have been described elsewhere11, and here we describe one of the specific methods that is currently used to microinject sea urchin (Strongylocentrotus purpuratus) newly fertilized eggs. For over a century, sea urchins have been a valuable experimental model15,16. Sea urchins are evolutionarily closely related to chordates (including us) and analysis of their genome revealed that they contain all the major gene families as the human17. They produce a large number of synchronously developing transparent embryos that can be easily manipulated. Using sea urchin as a model organism, the sea urchin community has contributed to our understanding of the fertilization process18-21, cell biological processes22-24, and the gene regulatory networks (GRNs)25-28.
Microinjection into sea urchin zygotes requires several steps. First, the eggs need to be immobilized prior to injections (described below). Microinjection dishes are coated with protamine sulfate (PS), which creates a positively-charged surface to which the negatively charged eggs can adhere3. The eggs are dejellied by incubation in acidic sea water (pH 5.15) for 10 min, followed by two washes in natural sea water or artificial sea water (pH 8.0). The dejellied eggs are carefully rowed in a straight line in the middle of PS-coated dish in the presence of 1 mM 3-aminotriazole (3-AT), which is required to inhibit the activity of ovoperoxidase that is secreted from the cortical granules of the egg as a result of fertilization29. This step is important to prevent hardening of the fertilization envelope and to facilitate microinjection needle entry. As an alternative to 1 mM 3-AT, 10 mM paraminobenzoic acid (PABA) can be used. The injection solution is loaded into a microinjection needle using specialized microloading pipette tip and mounted on a holder attached to micromanipulator and pressure unit (Figure 1). Each needle can be used to microinject individual zygotes in multiple experiments on separate days. Microinjection can be performed for 10-15 min until the zygotes harden. The zygotes are then washed with the sea water and cultured at 15 &#176;C. When the embryos reach hatching blastula stage, they release hatching enzyme that digests components of the fertilization envelope30 and allow them to naturally detach from the PS-coated dish. If necessary, the embryos can be gently detached from the dish using a mouth pipette or Pasteur pipette by gently blowing sea water onto the embryos. The described method enables efficient and reliable microinjection of 100-400 newly fertilized eggs on a single dish, providing a high-throughput method for downstream analyses.

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

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.

GFP and mCherry reporter constructs were in vitro transcribed and microinjected into the newly fertilized eggs. Embryos were incubated at 15 &#176;C for 24 hr (until the blastula stage) and imaged using Zeiss Observer Z1 microscope. Injection of reporter constructs did not lead to any developmental defects (Figure 6). For quantification of fluorescent signals, image acquisition was performed at low magnification (100X) to maximally capture fluorescent pixels (Figures 6D-F). Fluo...

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High Throughput Microinjections of Sea Urchin Zygotes

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 ...

high-throughput-microinjections-of-sea-urchin-zygotes

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Biology

14.4K visualizações.  University of Delaware.  A microinjecção é uma técnica comum usada para fornecer estruturas de ADN, ARNm, oligonucleidos morfolino anti-sentido ou outros tratamentos em ovos, embriões, e as células de várias espécies. 