Name: generating transgenics and knockouts in strongyloides species by microinjection
Uploaded: 2021-10-07T14:45:00
Description: Watch this Scientific Journal Video about Generating Transgenics and Knockouts in Strongyloides Species by Microinjection at JoVE.com

pPV540 and pPV402 were kind gifts from Dr. James Lok at the University of Pennsylvania. We thank Astra Bryant for helpful comments on the manuscript. This work was funded by a Burroughs-Wellcome Fund Investigators in the Pathogenesis of Disease Award, a Howard Hughes Medical Institute Faculty Scholar Award, and National Institutes of Health R01 DC017959 (E.A.H.).

NOTE: Gerbils were used to passage S.&#160;stercoralis, and rats were used to passage S.&#160;ratti. All procedures were approved by the UCLA Office of Animal Research Oversight (Protocol No. 2011-060-21A), which adheres to AAALAC standards and the Guide for the Care and Use of Laboratory Animals. The following tasks must be completed at least one day before microinjecting: worm culturing, preparing microinjection pads, creating constructs for the microinjection mix, and spreading bacteria (E. coli<...

<ol>
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This microinjection protocol details the methods for introducing constructs for transgenesis and CRISPR/Cas9-mediated mutagenesis into S.&#160;stercoralis and S.&#160;ratti. For both S.&#160;stercoralis and S.&#160;ratti, post-injection survival and the rate of transgenesis or mutagenesis are subject to several variables that can be fine-tuned.
The first critical consideration for successful transgenesis is how plasmid transgenes are constructed. Previous st...

Strongyloides stercoralis has long been overlooked as an important human pathogen compared to the more widely recognized hookworms and the roundworm Ascaris lumbricoides1. Previous studies of worm burden often severely underestimated the prevalence of S.&#160;stercoralis due to the low sensitivity of common diagnostic methods for S.&#160;stercoralis2. In recent years, epidemiological studies based on improved diagnostic tools have estimated that the true prevalence of S.&#160;stercoralis infections is much higher than previously reported, approximately 610 million people worldwide2.
Both S.&#160;stercoralis and other Strongyloides species, including the closely related rat parasite and common lab model S.&#160;ratti, have an unusual life cycle that is advantageous for experimental genomic studies because it consists of both parasitic and free-living (environmental) generations3 (Figure 1). Specifically, both S.&#160;stercoralis and S.&#160;ratti can cycle through a single free-living generation. The free-living generation consists of post-parasitic larvae that develop into free-living adult males and females; all progeny of the free-living adults develop into infective larvae, which must infect a host to continue the life cycle. Furthermore, this environmental or free-living generation can be experimentally manipulated in the laboratory. Because free-living Strongyloides adults and C. elegans adults share similar morphology, techniques such as intragonadal microinjection that were originally developed for C. elegans can be adapted for use with free-living adult Strongyloides4,5. While DNA is generally introduced into free-living adult females, both males and females of Strongyloides can be microinjected6. Thus, functional genomic tools are available to interrogate many aspects of the biology of Strongyloides. Other parasitic nematodes lack a free-living generation, and as a result, are not as readily amenable to functional genomic techniques3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63023/63023fig01.jpg" /> 
Figure 1: The Strongyloides stercoralis life cycle. The S.&#160;stercoralis parasitic females inhabit the small intestine of their mammalian hosts (humans, non-human primates, dogs). The parasitic females reproduce by parthenogenesis and lay eggs within the small intestine. The eggs hatch while still inside the host into post-parasitic larvae, which are then passed into the environment with feces. If the post-parasitic larvae are male, they develop into free-living adult males. If the post-parasitic larvae are female, they can either develop into free-living adult females (indirect development) or third-stage infective larvae (iL3s; direct development). The free-living males and females reproduce sexually to create progeny that are constrained to become iL3s. Under certain conditions, S.&#160;stercoralis can also undergo autoinfection, in which some of the post-parasitic larvae remain inside the host intestine rather than passing into the environment in feces. These larvae can develop into autoinfective larvae (L3a) inside the host, penetrate through the intestinal wall, migrate through the body, and eventually return to the intestine to become reproductive adults. The life cycle of S.&#160;ratti is similar, except that S.&#160;ratti infects rats and does not have an autoinfective cycle. The environmental generation is key to using Strongyloides species for genetic studies. The free-living adult females (P0) can be microinjected; their progeny, which will all become iL3s, are the potential F1 transgenics. This figure has been modified from Castelletto et al.3. <a href="https://www.jove.com/files/ftp_upload/63023/63023fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
S.&#160;stercoralis shares many aspects of its biology with other gastrointestinal human-parasitic nematodes, including host invasion and host immune modulation. For example, human-parasitic hookworms in the genera Necator and Ancylostoma also infect by skin penetration, navigate similarly through the body, and ultimately reside as parasitic adults in the small intestine7. Thus, many gastrointestinal nematodes likely use common sensory behaviors and immune evasion techniques. As a result, the knowledge gleaned from Strongyloides will complement findings in other less genetically tractable nematodes and lead to a more complete understanding of these complex and important parasites.
This microinjection protocol outlines the method for introducing DNA into Strongyloides free-living adult females to make transgenic and mutant progeny. The strain maintenance requirements, including the developmental timing of adult worms for microinjections and the collection of transgenic progeny, are described. Protocols and a demonstration of the complete microinjection technique, along with protocols for culturing and screening transgenic progeny, are included, along with a list of all necessary equipment and consumables.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(&#8722;)-Nicotine, &#8805;99% (GC), liquid</td>
			<td>Sigma-Aldrich</td>
			<td>N3876-5ML</td>
			<td>nicotine for paralyzing worms</td>
		</tr>
		<tr>
			<td>3&#34; iron C-clamp, 3&#34; x 2&#34; (capacity x depth)</td>
			<td>VWR</td>
			<td>470121-790</td>
			<td>C-clamp to secure setup to bench top</td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>Phenix</td>
			<td>RBA-500</td>
			<td>agarose for slides</td>
		</tr>
		<tr>
			<td>Bone char, 4 lb pail, 10 x 28 mesh</td>
			<td>Ebonex</td>
			<td>n/a</td>
			<td>charcoal for fecal-charcoal cultures</td>
		</tr>
		<tr>
			<td>Bone char, granules, 10 x 28 mesh</td>
			<td>Reade</td>
			<td>bonechar10x28</td>
			<td>charcoal for fecal-cultures (alternative to the above)</td>
		</tr>
		<tr>
			<td>Coarse micromanipulator</td>
			<td>Narishige</td>
			<td>MMN-1</td>
			<td>coarse micromanipulator</td>
		</tr>
		<tr>
			<td>Corning Costar Spin-X centrifuge tube filters</td>
			<td>Fisher</td>
			<td>07-200-385</td>
			<td>microfilter column</td>
		</tr>
		<tr>
			<td>Cover glass, 48 x 60 mm, No. 1 thickness</td>
			<td>Brain Research Lab</td>
			<td>4860-1</td>
			<td>coverslips (48 x 60 mm)</td>
		</tr>
		<tr>
			<td>Deep Petri dishes, heavy version with 6 vents, 100 mm diameter</td>
			<td>VWR</td>
			<td>82050-918</td>
			<td>10 cm Petri dishes (for fecal-charcoal cultures)</td>
		</tr>
		<tr>
			<td>Eisco retort base w/ rod</td>
			<td>Fisher</td>
			<td>12-000-101</td>
			<td>stand for Baermann apparatus</td>
		</tr>
		<tr>
			<td>Eppendorf FemtoJet microinjector microloader tips</td>
			<td>VWR</td>
			<td>89009-310</td>
			<td>for filling microinjection needles</td>
		</tr>
		<tr>
			<td>Fisherbrand absorbent underpads</td>
			<td>Fisher</td>
			<td>14-206-62</td>
			<td>bench paper (for prepping)</td>
		</tr>
		<tr>
			<td>Fisherbrand Cast-Iron Rings</td>
			<td>Fisher</td>
			<td>14-050CQ</td>
			<td>Baermann o-ring</td>
		</tr>
		<tr>
			<td>Fisherbrand tri-cornered polypropylene beakers</td>
			<td>Fisher</td>
			<td>14-955-111F</td>
			<td>Plastic beaker (for mixing)</td>
		</tr>
		<tr>
			<td>Fisherbrand tri-cornered polypropylene beakers</td>
			<td>Fisher</td>
			<td>14-955-111F</td>
			<td>Plastic beaker (for catch bucket/water bucket)</td>
		</tr>
		<tr>
			<td>Fisherbrand tri-cornered polypropylene beakers</td>
			<td>Fisher</td>
			<td>14-955-111F</td>
			<td>Plastic beaker (x2) (to make holder)</td>
		</tr>
		<tr>
			<td>Gorilla epoxie in syringe</td>
			<td>McMaster-Carr</td>
			<td>7541A51</td>
			<td>glue (to attach tubing)</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898-50ML</td>
			<td>halocarbon oil</td>
		</tr>
		<tr>
			<td>High-temperature silicone rubber tubing for food and beverage, 1/2&#34; ID, 5/8&#34; OD</td>
			<td>McMaster-Carr</td>
			<td>3038K24</td>
			<td>tubing (for funnel)</td>
		</tr>
		<tr>
			<td>KIMAX funnels, long stem, 60&#176; Angle, Kimble Chase</td>
			<td>VWR</td>
			<td>89001-414</td>
			<td>Baermann funnel</td>
		</tr>
		<tr>
			<td>Kimberly-Clark Professional Kimtech Science benchtop protectors</td>
			<td>Fisher</td>
			<td>15-235-101</td>
			<td>bench paper (for prepping)</td>
		</tr>
		<tr>
			<td>Leica stereomicroscope with fluorescence</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td>GFP stereomicroscope for identifying and sorting transgenic worms</td>
		</tr>
		<tr>
			<td>microINJECTOR brass straight arm needle-holder</td>
			<td>Tritech</td>
			<td>MINJ-4</td>
			<td>microinjection needle holder</td>
		</tr>
		<tr>
			<td>microINJECTOR system</td>
			<td>Tritech</td>
			<td>MINJ-1</td>
			<td>microinjection system</td>
		</tr>
		<tr>
			<td>Mongolian Gerbils</td>
			<td>Charles River Laboratories</td>
			<td>213-Mongolian Gerbil</td>
			<td>gerbils for maintenance of S. stercoralis, male 4-6 weeks</td>
		</tr>
		<tr>
			<td>Nasco Whirl-Pak easy-to-close bags, 18 oz</td>
			<td>VWR</td>
			<td>11216-776</td>
			<td>Whirl-Pak sample bags</td>
		</tr>
		<tr>
			<td>Nylon tulle (mesh)</td>
			<td>Jo-Ann Fabrics</td>
			<td>zprd_14061949a</td>
			<td>nylon mesh for Baermann holder</td>
		</tr>
		<tr>
			<td>Platinum wire, 36 Gauge, per inch</td>
			<td>Thomas Scientific</td>
			<td>1233S72</td>
			<td>platinum/iridium wire for worm picks</td>
		</tr>
		<tr>
			<td>Puritan tongue depressors, 152 mm (L) x 17.5 mm (W)</td>
			<td>VWR</td>
			<td>62505-007</td>
			<td>wood sticks (for mixing samples)</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td>QIAGEN miniprep kit</td>
		</tr>
		<tr>
			<td>Rats-Long Evans</td>
			<td>Envigo</td>
			<td>140 HsdBlu:LE Long Evans</td>
			<td>rats for maintenance of S. ratti, female 4-6 weeks</td>
		</tr>
		<tr>
			<td>Rats-Sprague Dawley</td>
			<td>Envigo</td>
			<td>002 Hsd:Sprague Dawley SD</td>
			<td>rats for maintenance of S. ratti, female 4-6 weeks</td>
		</tr>
		<tr>
			<td>Really Useful Boxes translucent storage boxes with lids, 1.6 L capacity, 7-5/8&#34; x 5-5/16&#34; x 4-5/16&#34;</td>
			<td>Office Depot</td>
			<td>452369</td>
			<td>plastic boxes for humidified chamber</td>
		</tr>
		<tr>
			<td>Shepherd techboard, 8 x 16.5 inches</td>
			<td>Newco</td>
			<td>999589</td>
			<td>techboard</td>
		</tr>
		<tr>
			<td>Stainless steel raised wire floor</td>
			<td>Ancare</td>
			<td>R20SSRWF</td>
			<td>wire cage bottoms</td>
		</tr>
		<tr>
			<td>StalkMarket compostable cutlery spoons, 6&#34;, white, pack of 1,000</td>
			<td>Office Depot</td>
			<td>9587303</td>
			<td>spoons</td>
		</tr>
		<tr>
			<td>Stender dish, stacking type, 37 x 25 mm</td>
			<td>Carolina (Science)</td>
			<td>741012</td>
			<td>watch glasses (small, round)</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Motic</td>
			<td>K-400 LED</td>
			<td>dissecting prep scope</td>
		</tr>
		<tr>
			<td>Storage tote, color clear/white, outside height 4-7/8 in, outside length 13-5/8 in, Sterilite</td>
			<td>Grainger</td>
			<td>53GN16</td>
			<td>plastic boxes for humidified chamber</td>
		</tr>
		<tr>
			<td>Sutter P-30 micropipette puller</td>
			<td>Sutter</td>
			<td>P-30/P</td>
			<td>needle puller with platinum/iridium filament</td>
		</tr>
		<tr>
			<td>Syracuse watch glasses</td>
			<td>Fisher</td>
			<td>S34826</td>
			<td>watch glasses (large, round)</td>
		</tr>
		<tr>
			<td>Thermo Scientific Castaloy fixed-angle clamps</td>
			<td>Fisher</td>
			<td>05-769-2Q</td>
			<td>funnel clamps (2x)</td>
		</tr>
		<tr>
			<td>Three-axis hanging joystick oil hydrolic micromanipulator</td>
			<td>Narishige</td>
			<td>MM0-4</td>
			<td>fine micromanipulator</td>
		</tr>
		<tr>
			<td>United Mohr pinchcock clamps</td>
			<td>Fisher</td>
			<td>S99422</td>
			<td>Pinch clamps (2x)</td>
		</tr>
		<tr>
			<td>Vented, sharp-edge Petri dishes (60 mm diameter)</td>
			<td>Tritech Research</td>
			<td>T3308P</td>
			<td>6 cm Petri dishes (for small-scale fecal-charcoal cultures)</td>
		</tr>
		<tr>
			<td>VWR light-duty tissue wipers</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>lining for Baermann holder</td>
		</tr>
		<tr>
			<td>watch glass, square, 1-5/8 in</td>
			<td>Carolina (Science)</td>
			<td>742300</td>
			<td>watch glasses (small, square)</td>
		</tr>
		<tr>
			<td>Whatman qualitative grade plain circles, grade 1, 5.5 cm diameter</td>
			<td>Fisher</td>
			<td>09-805B</td>
			<td>filter paper (for 6 cm Petri dishes)</td>
		</tr>
		<tr>
			<td>Whatman qualitative grade plain circles, grade 1, 9 cm diameter</td>
			<td>Fisher</td>
			<td>09-805D</td>
			<td>filter paper (for 10 cm Petri dishes)</td>
		</tr>
		<tr>
			<td>World Precision Instrument borosilicate glass capillary, 1.2 mm x 4 in</td>
			<td>Fisher</td>
			<td>50-821-813</td>
			<td>glass capillaries for microinjection needles</td>
		</tr>
		<tr>
			<td>X-Acto Knives, No. 1 Knife With No. 11 Blade</td>
			<td>Office Depot</td>
			<td>238816</td>
			<td>X-Acto knives without blades to hold worm picks</td>
		</tr>
		<tr>
			<td>Zeiss AxioObserver A1</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td>inverted microscope</td>
		</tr>
	</tbody>
</table>

generating transgenics and knockouts in strongyloides species by microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

If the experiment was successful, the F1 larvae will express the transgene and/or mutant phenotype of interest (Figure 4). However, transformation rates are highly variable and depend on the constructs, the health of the worms, the post-injection culturing conditions, and the skill of the experimenter. In general, a successful experiment will yield &#62;15 F1 larvae per injected female and a transformation rate of &#62;3% for fluorescent markers. If the total number of ...

Watch this Scientific Journal Video about Generating Transgenics and Knockouts in Strongyloides Species by Microinjection at JoVE.com

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Research

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