Name: microinjection wound assay and in vivo localization of epidermal wound response reporters in drosophila embryos.
Uploaded: 2013-11-01T21:20:00
Duration: 11 min 12 s
Description: Watch this Scientific Journal Video about Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos. at JoVE.com

This protocol has been developed in collaboration with previous members of the McGinnis Lab, Kim A. Mace and Joseph C. Pearson. We thank the continued efforts by the Bloomington Drosophila Stock Center for their work to organize and distribute valuable stocks.
This work was supported by funding from the National Institutes of Health (R01 GM077197 and K12 GM68524) and the family of Herbert Stern. MTJ is currently supported by Grant Number 5G12RR003060-26 from the National Center for Research Resources and Grant Number 8G12MD7603-27 from the National Institute On Minority Health and Health Disparities. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Minority Health And Health Disparities or the National Institutes of Health.

The Drosophila embryonic wound protocol can be summarized in five steps: (1) Embryo Collection, (2) Embryo Preparation (3) Embryo Wounding, (4) Embryo Microinjection, and (5) Embryo Fixation.
1. Embryo Collection
<ol>
	<li>Collect adult flies, 2-3 days after eclosing, add 50 females and 25 males to an embryo collection cage.</li>
	<li>Place a fresh apple juice agar plus yeast plate each morning for 3 days. 
	Notes: For optimal embryo collection, cycle the flies on a 12 hr diurnal schedule in a 25 &#176;C incubator (05:00 lights &#34;ON&#34; and 17:00 lights &#34;OFF&#34;); female flies respond to changes in the light cycle and will deposit larger quantities of eggs during a switch from lights &#34;ON&#34; to lights &#34;OFF&#34;10,11.</li>
	<li>In the afternoon of Day-3, place a fresh apple juice plus yeast agar plate on the cage and collect embryos for 2 hr. Place a new yeast plate on the cage and save the 2 hr collection plate.</li>
	<li>Store the collection plate for an additional 14&#160;hr&#160;at 25 &#176;C to age the embryos. 
	Notes: Wound healing can be assayed at any stage during Drosophila development; the wound-induced transcriptional reporters described in this paper have an optimal activity between stages 15-16 12. By late stage 17, the embryo is too old to easily pierce the maturing cuticle and efficiently activate the wound reporters or detect wound-induced transcription. To allow the collection time and the wounding time to occur during peak lab hours, we follow a schedule of embryo collection for 2 hr (16:00-18:00), exchange the collection plate with a new plate at 18:00, age the collection plate at 25 &#176;C for 14 hr (overnight), and wound the embryos at 08:00 (next day).</li>
</ol>
2. Embryo Preparation
Notes: This protocol includes the use of many tools and objects that are sharp or made of glass. Handle all sharps and glass objects with care to avoid personal injury.
<ol>
	<li>Gently remove embryos from collection plates with a paintbrush by adding 2-3 ml water to plate and swirling. Transfer water and embryos from plate to nylon mesh and collection tube.</li>
	<li>Add 4-5 ml of bleach to the cap of a plastic Petri dish plate and soak the mesh covered end of the collection tube containing the embryos in bleach for 2 min; which removes the outer eggshell of the embryo. Manually rotate the collection tube a couple of times to prevent embryos from clumping in the bleach. Rinse embryos with water 10x and dry the mesh covered collection tube containing the dechorionated embryos on a paper towel.</li>
	<li>Use a dissecting needle to transfer &#8764;50 embryos from nylon mesh to an agar plate. We add green food coloring to the agar plate to add contrast and aid in embryo alignment. Under a dissecting microscope, make two parallel rows of &#8764;25 embryos, aligning the embryos on the slide so that they will be at right angles to the puncture needle on the microinjection apparatus. Leave a little space (about 1 embryo length) between the embryos for gas exchange purposes. The two parallel rows should be approximately 5 mm apart.</li>
	<li>Place a slide with double stick tape on top of the aligned embryos and carefully press the slide to transfer embryos from the green plate to the slide. Then air dry the embryos &#8764;25 min to reduce internal pressure on the embryo and prevent a burst when wounding.</li>
	<li>Place 2-3 drops of halocarbon oil mix over the embryos to prevent further dehydration.</li>
</ol>
3. Embryo Wounding
<ol>
	<li>Place the embryo slide in the injection microscope stage. Find the embryos in the field of view and practice moving the stage of the inverted microscope. 
	Notes: Before working with the needle, focus on middle plane of embryo along the dorsoventral axis. To allow for efficient wounding of the aligned embryos, begin wounding the row closer to the needle apparatus. Move the stage to bring the top of the row of aligned embryos into the field of view.</li>
	<li>Carefully place and secure a pulled-needle in the needle holder. Use the micromanipulator to move the needle down toward slide. Align the needle with the embryo, in same focal plane. Once the needle is in focus with the embryo, do not move the needle with the micromanipulator.</li>
	<li>Move the stage to puncture the embryo through and through, then move to the next embryo in the row. When done wounding the first row, use the micromanipulator to move the needle completely up and away from the stage before moving the slide; manually rotate the slide 180&#176;&#160;and wound the second row.</li>
	<li>Store the embryos under halocarbon oil at room temperature and proceed with embryo fixation if doing in situ hybridization or antibody staining (see Procedure step&#160;5). Alternatively, store the embryos for 4-6 hr under halocarbon oil and proceed with wound transcriptional fluorescent reporter visualization (Representative Results). 
	Notes: For the visualization of the fluorescent reporter, we anesthetize the embryos using 50% 1-phenoxy-2-propanol13. The embryos are removed from the halocarbon oil and placed on a new slide. We place glass beads around the embryos, add a few drops of the anesthetic, and place a cover slip on the embryos.</li>
</ol>
4. Embryo Microinjection
Notes: The microinjection apparatus that we use in the protocol is not automated to deliver specific volume during the assay. We add a dye to the solution to visualize the microinjection and try to normalize the volume delivered. If too much solution is injected into the embryo, the vitelline membrane will burst.
<ol>
	<li>Load needle from the rear with 1 &#181;l of chemical solution plus dye (e.g. 0.6 M H2O2). Allow capillary action to draw the chemical solution to the tip of the needle.</li>
	<li>To break a needle, place a cover slip at an angle on a slide and coat the edge with halocarbon oil mix. Bring the mounted needle into focus with the edge of the angled cover slip and gently move cover slip edge across the tip of the needle until broken. Apply pressure on microinjection apparatus and check that the chemical solution plus dye is flowing out the needle.</li>
	<li>Place the slide containing aligned embryos on the microscope stage. Proceed to focus the embryo and the needle in the same plane. Simultaneously puncture and microinject chemical solution plus dye into each embryo. 
	Notes: Clogged needles occur frequently; break a new needle to avoid incomplete microinjection. A blunt needle tip does not wound as cleanly as an angled needle tip. Adjusting the coverslip to a non-90&#176; angle during breakage is optimal to produce an angled needle tip.</li>
</ol>
5. Formaldehyde Fixation
Notes: The chemicals used in this Drosophila fixation are harmful. Use personal protective equipment (e.g. gloves, laboratory coat, and safety goggles) when handling the chemicals. Follow individual institutional guidelines for disposal of regulated chemicals.
<ol>
	<li>After wounding, wait 15, 30, 60, or more minutes for the transcriptional activation of wound-induced genes to occur. To remove embryos from injection slides, carefully drain the halocarbon oil mix from the slides, lean the slide against a rack and blot the end with a Kimwipe.</li>
	<li>Use a glass pipette to rinse heptane over the slide and wash the rinse liquid into a glass Petri dish. Heptane will dissolve the tape and release the embryos. Continue to rinse until all the embryos are removed from the slide. Proceed to the next slide, until all embryos are removed from slides.</li>
	<li>Transfer the heptane plus embryo solution from the glass dish into a 20 ml scintillation vial. Shake the vial for 2-3 min, remove the heptane and add 5 ml of fresh heptane.</li>
	<li>Add 5 ml of fresh, fixation solution (see reagents list for recipe). Cap the vial and attach to an orbital platform shaker. Shake the vial containing the embryos for 25 min at 220-230 rpm.</li>
	<li>After shaking, let the bubbles at the interface pop. Completely remove the bottom, aqueous phase with a pipette, avoiding pulling up the embryos. The bottom layer consists of the fixation solution and should be disposed properly.</li>
	<li>Add 5 ml of methanol, cap the vial and shake vigorously by hand for 20-30 sec, swirl and place it on the bench. Wounded embryos will stay at the interphase and will not sink to the bottom. 
	Notes: After fixation the vitelline membrane of an unwounded embryo will normally burst during the dehydration step with methanol. However, the vitelline membrane in a wounded embryo is already broken and the dehydration step will not remove the vitelline membrane. Hand-devitellinzation is required to complete the fixation protocol.</li>
	<li>Carefully remove the top heptane layer and then add 5 ml of fresh methanol. Shake the vial and the embryos should sink.</li>
	<li>Remove the methanol plus embryo solution with a glass transfer pipette and collect in a nylon mesh collection tube in a glass dish.</li>
	<li>Remove the methanol and rinse the embryos with 1x&#160;PBS solution.</li>
	<li>Transfer the embryos from the nylon mesh to an apple juice plate. Spread the embryos out along the surface of the plate.</li>
	<li>Transfer the embryos to a double stick tape glass slide. Cover the embryos with 1x&#160;PBS. 
	Notes: The embryos tend to clump because of the vitelline membrane. Using an apple juice place allows for quick separation of the clumped embryos. The embryos must be removed from the plate and transferred to a double stick tape glass slide because embryos sink into the agar plate when force is applied to remove the embryo from the vitelline membrane.</li>
	<li>Carefully push the embryos with a dissecting needle to &#34;pop&#34; the vitelline membrane. The embryo should sink in the PBS.</li>
	<li>Use a glass pipette to transfer the 1x&#160;PBS plus embryos to a 1.5 ml tube. Rinse the embryos with 1x&#160;PBS and store at 4 &#176;C. Proceed to dehydrate the embryos using an Ethanol:PBS series and continue with in situ hybridization or immunolocalization protocol14.</li>
</ol>

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No conflicts of interest declared.

An immediate transcriptional regulatory response to external cues allows cells to coordinate a localized response to extracellular stimuli, which can include stress, damage, or infection. The improper control of a localized response can have damaging effects on neighboring cells, as well as a waste of resources to repair the injury. The Drosophila embryo provides an excellent system to conduct basic wound healing experiments in an inexpensive and easy-to-maintain model organism. The potential for collaborations between research in other model organisms and Drosophila provides a unique opportunity to explore many biological questions.
An additional technique of the wound reporters is the genetic combination of mutant backgrounds, providing an efficient method for testing the contribution of new genes to the activation of the epidermal wound response pathway. We have epidermal wound-induced transcriptional reporters available on both the 2nd and 3rd chromosomes5.&#160;In this study we use UAS and GAL4 systems of overexpression16. For studies with lethal mutant alleles we determine the genetic background using a fluorescent balancer chromosome, e.g. Kruppel-GFP17. We have analyzed the wound reporter localization in several genetic backgrounds (Table1).
A possible modification of the technique is to combine microinjection and wounding. Microinjection can be used to introduce a chemical solution into the embryo. Several chemical compounds have been tested and were found to regulate the activity of the epidermal wound reporters9. This method of simultaneous wounding and microinjection can be useful to increase the number of cells in the Drosophila embryo that respond a wound signal and can be directly used to monitor gene expression changes after wounding18. A future application of the microinjection technique will be to test novel chemicals for regulation of the epidermal wound response.
Drosophila as a model system for genetic studies offers a wide range of simple protocols to efficiently address complex questions. Recent reports on the feasibility of Drosophila techniques highlights the use of hemocyte migration19 and parasitic wasp infection20 as a means to further broaden the impact of the Drosophila research community. In addition to wound repair studies, Drosophila provides a classical model for regeneration with the imaginal disc system21,22. The combined advances in imaginal disc regeneration studies and puncture/microinjection wounding provides an excellent system to discover well-conserved components of tissue repair.

Manipulation of Drosophila embryos using a microinjection technique is a well-established assay1. The significance of the epidermal wound response reporter method is to combine the protocol for puncture/microinjection with fluorescent reporters and visualize an in vivo response to epidermal injury in Drosophila embryos. The goal of this method is to enable a broader set of scientists to use Drosophila as a tool to investigate the processes that regulate the transcriptional response to epidermal puncture injury. The single-layered epidermis in Drosophila provides a simple system to study an epidermal wound response after a puncture injury2. The Drosophila embryo is a robust model system to genetically dissect the stages of wound repair, including wound response, inflammation, and reepithelialization3. This is in part because many or most zygotic mutants survive to the end of embryogenesis and develop epidermal barriers even when developmental patterning goes profoundly awry. Previous characterization of a well-conserved transcription factor Grainy head (Grh), identified that Grh-target genes Dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple) are transcriptionally activated around the sites of injury4. Drosophila as a model organism for wound response provides a complementary line of investigation to advance studies in mammalian wound healing5. Subsequent studies have identified additional Drosophila wound-induced genes and developed a &#34;toolkit&#34; of many fluorescent wound reporters to monitor the in vivo epidermal wound response to clean wounding6. Recent reports on the regulation of Drosophila wound healing have focused on the wound closure phenotype, allowing discovery of many pathways regulating cell migration7,8. With the analysis of the fluorescent wound reporters, our studies have identified a novel set of genes required for the localized expression of epidermal wound-inducible genes9. One of the merits of the wound response reporter method is that the results provide more insight into a mechanism of how signals are transduced from the site of injury to the neighboring cells. However, one of the demerits of the wound response reporter method is that the results do not directly link to phenotypes in wound healing or repair. Broader use of the clean wound puncture protocol in genetic and chemical screens will allow new regulators of the transcriptional response to epidermal wounding to be identified&#160;and further the translation of wound healing discoveries into mammalian models of injury treatments.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">yeast</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:133px;">51475</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apple juice</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">agar</td>
			<td>Fisher Scientific</td>
			<td>50-824-297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bleach</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 700 W</td>
			<td>Sigma Aldrich</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 27 W</td>
			<td>Sigma Aldrich</td>
			<td>H8773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-phenoxy-2-propanol</td>
			<td>Sigma Aldrich</td>
			<td>484423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>H324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methyl-&#946;-cyclodextrin</td>
			<td>Sigma Aldrich</td>
			<td>C4555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">toluidine blue</td>
			<td>Sigma Aldrich</td>
			<td>89640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formaldehyde, 16%</td>
			<td>Polysciences, Inc</td>
			<td>18814-20</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heptane</td>
			<td>Fisher Scientific</td>
			<td>H360-1</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-1</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>Fisher Scientific</td>
			<td>50-899-90013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EGTA</td>
			<td>Fisher Scientific</td>
			<td>50-255-956</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RNase free H2O</td>
			<td>Fisher Scientific</td>
			<td>BP2819-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:121px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila incubator</td>
			<td>Genessee Scientific</td>
			<td>59-197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fly cage</td>
			<td>Genessee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">embryo collection tube</td>
			<td>Genessee Scientific</td>
			<td>46-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic Petri dish</td>
			<td>Fisher Scientific</td>
			<td>50-202-037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double-stick tape</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissection needle</td>
			<td>Fisher Scientific</td>
			<td>08-965A</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass cover slip</td>
			<td>Thermo Scientific</td>
			<td>3306</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope slide</td>
			<td>Thermo Scientific</td>
			<td>4445</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">capillary needle</td>
			<td>FHC</td>
			<td>30-30-1</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">needle puller</td>
			<td>Narishige</td>
			<td>PC10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microinjection needle holder</td>
			<td>Narishige</td>
			<td>MINJ4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micromanipulator</td>
			<td>Narishige</td>
			<td>MN151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted microscope</td>
			<td>Carl Zeiss</td>
			<td>Primo-Vert</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747A</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>22-063-172</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">transfer bulb</td>
			<td>Fisher Scientific</td>
			<td>03-448-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">scintillation vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-7</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">14-259-260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal</td>
			<td>Leica</td>
			<td>SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fixation solution</td>
			<td>(5 ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% formaldehyde</td>
			<td>2.5 ml</td>
			<td></td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>0.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EGTA</td>
			<td>0.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RNase free H2O</td>
			<td>1.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halocarbon oil mix</td>
			<td>(10 ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 700 W</td>
			<td>5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 27 W</td>
			<td>5 ml</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

microinjection wound assay and in vivo localization of epidermal wound response reporters in drosophila embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

To obtain the results shown in this method, the open reading frame of green fluorescent protein (GFP) is fused to a promoter/wound-induced enhancer sequence from Dopa Decarboxylase (Ddc) and DsRed is fused to a wound enhancer sequence from tyrosine hydroxylase (ple)4,6. In a live Drosophila embryo, maturation of the fluorescent wound reporter protein is optimal 4-6 hr after wounding, which allows time for the accumulation of sufficient protein to visualize, as well as time for the fluorescent protein to oxidize to the fluorescent state15. To observe the reporter localization, a compound fluorescent microscope is sufficient. To observe a higher magnification of the reporter localization, a confocal microscope is optimal to generate a maximum projection of multiple optical sections (Figure 1). Confocal in vivo imaging of Drosophila embryos is complicated by the rapid undulations of the embryo. . A full-view of embryo can be obtained with 20X objective and a close-up view of wound site can be obtained with 40X objective. A top-view of epidermal wound reporter localization can be imaged with 10 successive 1&#181;m optical sections. A lateral-view of epidermal wound reporter localization can be imaged with 40 successive 1 &#181;m optical sections.
Using standard genetic crossing methods, it is possible to combine wound reporters with genetic mutations. One example presented in this paper is Flotillin-2 (Flo-2), because loss-of-function (null allele generated with P-element insertion) and gain-of-function (overexpression generated with ubiquitous expression in all cells) mutants of Flo-2 demonstrate the Flo-2 gene product is necessary and sufficient to inhibit the localization of the Ddc-GFP wound reporters9 (Figures 2A and 2B). Using the microinjection protocol, it is possible to test whether introduction of chemical solutions superactivates or inhibits the localization of wound reporters. Chemically-wounded embryos were simultaneously wounded and injected with a 1:4 ratio of 1% toluidine blue dye and solubilized compounds. Toluidine blue dye allowed for visual confirmation of solubilized compounds being injected into the body cavity. Control embryos were wounded with a broken needle containing 1:4 ratio of 1% toluidine blue dye and solute without chemical. Two examples presented in this paper are hydrogen peroxide (H2O2) and methyl-&#946;-cyclodextrin (M&#946;CD), because both chemical solutions are sufficient to globally activate the localization of the Ddc-GFP wound reporters9 (Figures 2C and 2D). Hydrogen peroxide (H2O2) was diluted in H2O to 0.6 M. Methyl-&#946;-cyclodextrin (M&#946;CD) was solubilized in 1 mM NaOH to 3 mM. Using the embryo fixation protocol, it is possible to detect the transcription activation of wound response genes in a localized domain, surrounding a wound site. One example presented in this paper is the in situ hybridization of RNA probes to detect Ddc and ple transcripts4,6,9 (Figures 3A and 3B).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50750/50750fig1.jpg" width="600px" /> 
Figure 1. Ddc-GFP and ple-DsRed wound reporter localization. Brightfield and fluorescent images in wounded wild type embryos. (A) Top-view of Ddc-GFP wound reporter. (B) Top-view of ple-DsRed wound reporter. All images were collected on a Leica SP2 confocal microscope, with 20X objective. Arrows mark site of wound. Dashed lines in the data panels mark the outlines of embryos. <a href="https://www.jove.com/files/ftp_upload/50750/50750fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50750/50750fig2.jpg" width="500px" /> 
Figure 2. Localization of the Ddc-GFP wound reporter in genetic mutant backgrounds and chemical&#160;microinjected embryos. Brightfield and fluorescent images of wounded Flotillin-2 (Flo-2) mutant embryos. (A) Loss-of-function Flo-2 {KG00210} mutants, expansion of reporter activity throughout all epidermal cells. (B) Gain-of-function Flo-2 (armadillo-GAL4, UAS-Flo2) mutants, inhibition of reporter activity throughout all epidermal cells. (C) Hydrogen peroxide (H2O2) solution microinjection, expansion of reporter activity throughout all epidermal cells. (D) Methyl-&#946;-cyclodextrin (M&#946;CD) solution microinjection, expansion of reporter activity throughout all epidermal cells. All images were collected on a Leica SP2 confocal microscope, with 20X objective. Arrows mark site of wound. Dashed lines in the data panels mark the outlines of embryos. <a href="https://www.jove.com/files/ftp_upload/50750/50750fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50750/50750fig3.jpg" width="600px" /> 
Figure 3. Detection of wound response gene RNA transcripts. Fluorescent images of in situ hybridization and RNA detection in wounded wild type embryos. Ddc (A) and ple (B) RNA transcripts accumulate around the wound site. All images were collected on a Leica SP2 confocal microscope, with 20X objective. Arrows mark site of wound. Dashed lines in the data panels mark the outlines of embryos. <a href="https://www.jove.com/files/ftp_upload/50750/50750fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Table 1" fo:content-width="6in" src="/files/ftp_upload/50750/50750table1.jpg" width="600px" /> 
Table 1. Summary of epidermal wound response reporters. Insertions of the four wound response reporters (Ddc, ple, msn, kkv) are available on both the second and third chromosomes. All of the wound response reporters activate the fluorescence gene (GFP or DsRed) in a limited number of epidermal cells surrounding the site of puncture injury in the wild type background (e.g. &#34;local&#34; phenotype). Ddc and msn wound response reporters require Grh for activation of the fluorescence gene after puncture injury (e.g. &#34;none&#34; phenotype). All of the wound reporters require Flo-2 for localization of the fluorescence gene after puncture injury (e.g. &#34;global&#34; phenotype). <a href="https://www.jove.com/files/ftp_upload/50750/50750table1highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos. at JoVE.com

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

microinjection-wound-assay-vivo-localization-epidermal-wound-response

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...