This work was supported by the National Science Foundation IOS 0844948 and IOS 1024811

Keep all sea water or artificial seawater (SW), adult animals, and cultures at 15 &#176;C as much as is practical. Ensure eggs and zygotes are kept immersed in SW.
Commercially prepared sea salts reconstituted with distilled or reverse osmosis water serves well as a source of SW. Check salinity using a hydrometer and adjust salts or water to achieve optimum levels. Keep specific gravity levels between 1.020 to 1.025. Keep all glassware and plasticware separate from all other labware to avoid a...

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There are two critical steps that are difficult for novice users of this technique but are essential for successfully creating morphant embryos. The first is selecting healthy oocytes that will mature and fertilize properly. The percentage of normal development in a culture depends on the season, the health of the animal, and the number of times that oocytes have been harvested from a single individual. Oocytes tend to be of better quality from April through October. It is important to look carefully at the oocytes befor...

The sea star, Patiria miniata, (commonly known as the bat star) is emerging as an interesting and versatile model system for a variety of cellular1-3, developmental4,5, evolutionary6-8, and ecological studies9-11. Adult P. miniata are distributed along the pacific coast from Sitka, Alaska to Baja, California12 and are readily maintained in marine aquaria. Oocytes are obtainable year round and each female can shed tens of thousands of eggs. Oocytes are easily matured and fertilized externally13. The resulting embryos are transparent allowing for easy observation; they develop synchronously, and require only sea water for development. Whole genome assembly and multiple transcriptomes are also available for P. miniata (<a href="http://www.echinobase.org/Echinobase/">Echinobase.org</a>). Such advantages make them ideal for a range of research and teaching purposes.
In recent years, P. miniata has become a model system for developmental gene regulatory network analyses14-16. The aim of such studies is to identify the entire compliment of regulatory genes and determine the network of their interactions. Much of this work entails perturbing gene expression through introduction of antisense oligonucleotides or in vitro synthesized mRNAs. Additionally, cis regulatory analyses are used to characterize the function of regulatory DNA15. These analyses require introduction of perturbation reagents and/or DNA reporter constructs into embryos. Furthermore, to characterize the downstream effects of these perturbations, one must assay many embryos for changes in gene expression of potential targets. Techniques for microinjection of many hundreds of zygotes are central for this work.
Echinoderms, including P. miniata, require many months to reach sexual maturity. Because of this, it is generally not practical to develop and maintain transgenic lines of these animals for experimentation. Therefore, breeding of transgenic adults cannot efficiently create modified embryos. Instead, perturbation must occur de novo through microinjection. Microinjection offers an opportunity to modify embryos with reagents that are not cell-permeable. The following protocol describes a method to introduce DNA, mRNA, cell tracers, and morpholino antisense oligonucleotides into hundreds of fertilized eggs in one 2-3 hr sitting through microinjection. This produces sufficient material for a variety of downstream experiments including, but not limited to, qPCR, in situ hybridization, RNA-Seq, and western blotting.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1-Methyladenine</td>
			<td style="width:147px;">Acros Organics (Fisher Scientific)</td>
			<td style="width:119px;">AC20131-1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">190 micron nitex nylon filter</td>
			<td style="width:147px;">Small Parts (originally Sefar)</td>
			<td style="width:119px;">CMN-0185-C/5PK-05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">100 micron nitex nylon filter</td>
			<td style="width:147px;">Small Parts (originally Sefar)</td>
			<td style="width:119px;">CMN-0105-C/5PK-05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Polystyrene Petri Dishes, 60mm x 15mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Capillary tubing</td>
			<td style="width:147px;">FHC, Inc</td>
			<td style="width:119px;">30-30-0</td>
			<td style="width:167px;">For pulling microinjection needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Model P-97 Needle Puller</td>
			<td style="width:147px;">Sutter Instruments</td>
			<td style="width:119px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">Dextran, Rhodamine Green</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">D7163</td>
			<td style="width:167px;">If injecting a GFP expression reporter, it is helpful to substitute Texas Red dextran as an injection tracer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:147px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td style="width:167px;">Also available in many pet stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader Tips</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5242 956.003</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a method for microinjection of patiria minata zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

The goal of this protocol is to introduce reagents into embryos. We demonstrate the effectiveness of the protocol by injecting a DNA reporter construct that drives the expression of green fluorescent protein (GFP). Injected embryos express GFP in clonal patches (Figure 4A-B) as the DNA incorporates during early cleavage. Many reagents that are desirable to introduce into embryos are toxic in high quantities and to suboptimal batches of embryos. Toxicity manifests by delaying development, arresting develo...

Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

a-method-for-microinjection-of-patiria-minata-zygotes

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JoVE Journal

Biology

10.9K visualizações.  Carnegie Mellon University.  Métodos que produzem embriões morphant são essenciais para estudar mecanismos de desenvolvimento e redes de regulação gênicas. A estrela do mar Patiria miniata é um sistema modelo emergente para estes estudos. Aqui apresentamos um protocolo para a obtenção de gametas, produzindo culturas de embriões, e microinjeção rápida de zigotos de esta espécie. 