Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

Podsumowanie

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. stercoralis and S. ratti.

Streszczenie

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S. stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S. ratti is closely related to S. stercoralis and is often used as a laboratory model for S. stercoralis. Both S. stercoralis and S. 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.

Wprowadzenie

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. stercoralis due to the low sensitivity of common diagnostic methods for S. stercoralis². In recent years, epidemiological studies based on improved diagnostic tools have estimated that the true prevalence of S. stercoralis infections is much higher than previously reported, approximately 610 million peo....

Protokół

NOTE: Gerbils were used to passage S. stercoralis, and rats were used to passage S. 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 HB101) onto 6 cm Nematode Growth Media (NGM) plates⁸. The free-living females require a minimum of 24 h post-fecal collec....

Wyniki

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 >15 F₁ larvae per injected female and a transformation rate of >3% for fluorescent markers. If the total number of .......

Dyskusje

This microinjection protocol details the methods for introducing constructs for transgenesis and CRISPR/Cas9-mediated mutagenesis into S. stercoralis and S. ratti. For both S. stercoralis and S. 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.......

Materiały

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