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 ...

Using microinjection to deliver DNA constructs into Strongyloides allows us to generate knockouts and transgenics, which can be used to study how these worms develop, navigate their environment, and parasitize hosts. So far, this is the only successful technique for generating knockouts and transgenics in these parasites. One day before microinjection, add 180 microliters of the agarose solution onto a cover slip using a glass Pasteur pipette, then immediately drop a second cover slip on top to flatten the agarose into a thin pad.After 5 to 10 seconds, remove the top cover slip by sliding the two apart and determine which slide the agar pad is on, and lay it face up. Then, select a tiny piece of glass shard from a broken cover slip, and using forceps, gently press it into the agar near the top edge of the pad. On the day of the microinjection, line the Baermann holder, which is a sieve made from two plastic rings with two layers of nylon tuile netting secured between them with three overlapping pieces of lab tissue.Then add the fecal-charcoal mixture to the Baermann holder. Next place the Baermann holder with the fecal-charcoal mixture in the funnel, fold the pieces of tissues around the fecal-charcoal mix, and add enough water to submerge most of the fecal charcoal. Then, top the funnel with a 15-centimeter plastic Petri dish lid to contain the odor and label the funnel as needed.After one to two hours, hold a 50-milliliter centrifuge tube under the rubber tubing at the bottom of the funnel and carefully open the clamps at the bottom to dispense 30 to 40 milliliters of water containing worms into the 50-milliliter tube.Then. transfer 15 milliliters of the Baermann water containing the worms to a 15-milliliter centrifuge tube. Spin the tube, remove up to 13 milliliters of the supernatant, and discard it into a liquid waste container with iodine to kill any worms.After collecting all the worms, inspect the pellet of worms at the bottom of the tube. If no worms are visible, wait for one to two hours then collect more worms from the Baermann apparatus. Transfer the worms in as little water as possible to a six-centimeter 2%nematode growth medium plate with a lawn of E.coli HB101 and use this plate as the source plate for the microinjection.Under the dissecting microscope, cover the shard of glass on the microinjection pad cover slip with halocarbon oil using a standard platinum worm pick. Then, place the microinjection pad cover slip on the microinjection scope and locate the shard of glass covered in oil. Align the glass shard such that an edge is perpendicular to the direction of the needle to serve as the surface used to break the needle.Next, using a dissecting microscope, ensure that the needle has no bubbles or debris in the tapered shaft, then secure the needle 1 to 1.5 centimeters into the pressurized holder. Position the tip of the needle in the center of the microscope field-of-view by eye and under low-magnification, position the tip of the needle in the field-of-view perpendicular to the side of the glass shard. Then, switch to higher magnification and align the tip of the needle with the edge of the glass, near, but not touching it.Next, to break the tip of the needle and allow liquid flow, gently tap the needle on the side of the piece of glass while applying continuous pressure from the gas. Once the liquid begins to flow, check the shape of the tip and ensure that it is sharp with easily flowing liquid. When the liquid is flowing well from the needle, move the microinjection slide to the dissecting scope and add one to two microliters of halocarbon oil on the agar pad for placement of the worms.Transfer 20 to 30 young adult Strongyloides to a 2%nematode growth medium plate without bacteria for at least five minutes to remove excess surface bacteria and select single worms for microinjection. Add more worms to the plate as needed while injecting. Using a small amount of halocarbon oil on a worm pick, select a Strongyloides young adult female with one to four eggs in her gonad from the plate without bacteria and transfer the worm into a tiny drop of oil on the agar pad.Using the worm pick, gently position the worm so it is not coiled and the gonad is visible and easy to access. Then, position the worm in the microinjection microscope field-of-view, ensuring that the gonad is on the same side as the needle and positioned so that the needle will contact the gonad at a slight angle. Next, bring the tip of the needle to the side of the worm in the same focal plane.Then, aiming for the gonad arm near the middle of the worm, gently insert the needle into the gonad using a microinjector. Immediately apply pressure to the needle to gently fill the entire gonad arm with the DNA solution. After enough fluid has been injected, remove the needle and observe that the wound closes.Repeat the procedure with the other arm of the gonad, if it is visible. Once the injection is completed, quickly verify that the needle is not clogged by applying pressure with the tip of the needle on the agar pad, then transfer the slide with the injected worm to the dissecting microscope. To recover the injected worm, first place a few drops of worm-buffered saline on the worm to float it off the agar pad.Then collect a small amount of bacteria on a worm pick and touch the worm with the adherent bacteria to remove it from the liquid. Gently transfer the worm to the recovery plate. For behavioral experiments, transfer 20 to 30 larva to a 2%nematode growth medium plate with a thick HB101 bacteria lawn, and under a fluorescence dissecting microscope, identify the larvae expressing the transgene of interest.Then, using a worm pick, select the transgenic larvae and place them in a small watch glass with worm-buffered saline. For microscopy, using a razor blade, score a grid onto the plastic bottom of a 10-centimeter chemotaxis plate to easily track the location of the worms on the plate. Next, add three microliters of larvae in worm-buffered saline into a square on the grid, filling as many squares as needed.Then add 15-to 20-microliter drops of 1%nicotine in water to the worm drops. After four minutes, when the worms are paralyzed, screen them using a fluorescence dissecting microscope. Transgenic Strongyloides stercolaris larvae with an incomplete and complete act-2 mRFPmars expression pattern are show here.The patchy expression in the body wall muscle is more common when the transgene is not integrated into the genome. In contrast, consistent expression throughout the body wall muscle often indicates that the transgene has integrated into the genome. The key step is positioning the needle in the same plane of focus as the gonad to ensure the DNA is injected into the gonad and will be incorporated into the developing eggs.The development of this technique made it possible to study gene function in parasitic nematodes, which is providing new insights into mechanisms of parasitism and host-parasite interactions.

generating-transgenics-knockouts-strongyloides-species

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies&#8212;microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...