The authors acknowledge Channa Aluvihare and Yonus Gebermicale, ITF, UMD, for insight and support during the initial phase of protocol development. Tungsten needles were a generous gift from David O&#39;Brochta, ITF, UMD. We are thankful to Dr. Ladislav Simo for testing this protocol in I. ricinus and for insightful discussions. This project was funded by NIH-NIAID R21AI128393 and Plymouth Hill Foundation, NY to MG-N, startup funds from the University of Nevada to AN, the National Science Foundation Grant No. 2019609 to MG-N and AN, and a Peer-to-Peer Grant from IGTRCN to AS.

Ixodes scapularis adults were either purchased from Oklahoma State University (OSU) or reared at the University of Nevada, Reno (UNR) (IACUC protocol #21-001-1118).
1. Preparation of female ticks for embryo collection
NOTE: To collect eggs of appropriate age, it is important to synchronize egg-laying. Although egg-laying cues in ticks remain unclear, under the standard insectary conditions (27 &#176;C temperature and &#62;90% relative humidity (RH)), I. scapularis females begin laying eggs approximately 8 days post-host detachment. This timeline can be lengthened by storing replete females at 4 &#176;C. We have stored blood-fed females at 4 &#176;C up to 8 weeks without any negative effect on egg-laying. These conditions may need to be modified for each insectary.
<ol>
	<li>Store all replete females at 4 &#176;C with &#62;90% RH in a 6-quart plastic box lined with moist filter paper until used for microinjections.</li>
	<li>A week before injections, transfer females from 4 &#176;C to a 27 &#176;C incubator for the initiation of egg-laying.</li>
	<li>When the females start laying eggs (3-4 days after moving them to 27 &#176;C), remove any eggs using a fine-tipped paintbrush and prepare females for Gen&#233;&#39;s organ (wax gland) ablation. 
	NOTE: The Gen&#233;&#39;s organ extends from the area between the scutum and capitulum (dorsal base of the mouthparts), mouthparts bend over the ventral body surface (parallel to the surface), and the extended Gene&#39;s organ coats eggs as soon as the eggs are laid (Figure 1). Eggs laid before the Gen&#233;&#39;s organ removal or emptying of wax will have a wax coating and are not suitable for injections and must be discarded.</li>
	<li>To either remove or empty the Gen&#233;&#39;s organ, use clay to position the engorged female on a glass slide under a microscope oriented so&#160;that mouthparts are visible on both ventral and dorsal sides (Figure 1B,C).
	<ol>
		<li>Carefully poke the area exactly between the scutum and mouthparts using fine forceps and tungsten needles made in the lab using tungsten wire following a previously published protocol16 (needles can also be purchased commercially, and attached to a micro dissecting probe, see Table of Materials).</li>
		<li>Working the way inside with the tungsten needle, pull the wax gland out (Figure 1D,E). It may take several minutes to remove the gland. Wipe away any liquid wax secretion using lab wipes. 
		NOTE: The gland can also be removed from the ventral side right below the mouthparts; by reaching in the back towards scutum with a needle and forceps. Application of silicon glue around mouthparts also blocks Gene&#39;s organ eversion and can be used instead of removing or emptying the wax (personal communication with Dr. Ladislav Simo, INRAE, France). Engorged ticks will not stay on double-sided tape. Using clay to &#34;swaddle&#34; the tick helps keep them in place during manipulation. Use forceps to hold the tick and tungsten needle for dissection. The ventral part is easier to pierce with a needle and preferred by the authors.</li>
	</ol>
	</li>
	<li>To empty the gland, arrange the gravid tick as mentioned in step 1.4. Carefully puncture the area behind the mouthparts on the dorsal side and apply pressure on the ventral side using forceps. 
	NOTE: Alternatively, it&#160;is not necessary to remove the wax gland; emptying the gland of wax will last for several days. Egg-laying females have a white area around the mouth parts that indicates the location of the wax gland and can be used as a reference.
	<ol>
		<li>Place the edge of a lint-free lab wipe and remove the liquid wax coming out of the puncture site. Ensure to avoid hemolymph secretion, which is a clear liquid compared to slightly yellowish viscous wax. It may take 5-10 min until most of the wax is removed.</li>
	</ol>
	</li>
	<li>After manipulation, place females in an incubator at 27 &#176;C (&#62;90% RH) and allow them to recover for 1-2 days. 
	NOTE: Treated females usually take 1-2 days to recover and start laying eggs again.</li>
	<li>When the females begin laying eggs again, use a fine paintbrush to collect freshly deposited eggs (0-18 h post-egg-laying) and place them in a 1.5 mL microcentrifuge tube. 
	&#8203;NOTE: If using eggs from the same female over time, this procedure must be repeated after 4-5 days of egg-laying if glands were not removed.</li>
</ol>
2. Embryo treatment for microinjections
<ol>
	<li>Add ~200 &#181;L (or sufficient volume to cover the eggs, dependent on the number of eggs) of 5% (weight/volume) benzalkonium chloride/water (see Table of Materials) in the tube containing 0-18 h old eggs. Gently swirl the eggs with the paintbrush for 5 min to avoid the eggs settling into the bottom of the tube (for details, see Sharma et al.4). Remove the supernatant using a micropipette, leaving the eggs behind in tube. 
	CAUTION: Benzalkonium chloride is corrosive to skin and eyes, wear gloves and eye protection.</li>
	<li>Add ~200 &#181;L of distilled (DI) water to the tube containing eggs, swirl with a paintbrush, and remove the water from the microcentrifuge tube using a micropipette. Repeat the washing with DI water one more time.</li>
	<li>After washing with DI water, add ~200 &#181;L of 5% (w/v) sodium chloride (NaCl) and gently swirl with a paintbrush for 5 min. Remove the solution from the tube after 5 min and wash the eggs twice with DI water.</li>
	<li>Add ~100 &#181;L of 1% (w/v) NaCl solution in the microcentrifuge tube containing eggs. Keep the eggs in 1% (w/v) NaCl solution until they are used for injections.</li>
	<li>Start microinjections after about an hour. This timeline helps in proper desiccation of the eggs and prevents them from bursting. 
	&#8203;NOTE: Eggs can be kept in 1% (w/v) NaCl solution for 7-8 h without significantly affecting&#160;survival. If the eggs are difficult to inject or burst, then the eggs are not desiccated enough and can be further treated with 5% (w/v) NaCl for an additional 5 min.</li>
</ol>
3. Preparation of injection needles
<ol>
	<li>Insert an aluminosilicate capillary glass (with filament, see Table of Materials) into the needle puller following the manufacturer&#39;s instructions.</li>
	<li>Adjust the needle puller to the following settings: Heat: 575, Pull: 20, Velocity: 50, time: 200, and Pressure: 700.</li>
	<li>Activate the pull function of the needle puller and repeat the process for additional needles.</li>
	<li>Store the pulled needles by sticking them into lines of modeler&#39;s clay in a clean Petri dish.</li>
	<li>Load the needle with the injection mixture using a micro loader tip (see Table of Materials). 
	&#8203;NOTE: The comparison of needles used for tick and mosquito embryo injection is shown in Figure 2.</li>
</ol>
4. Slide setup for the microinjections
<ol>
	<li>Using double-sided tape, adhere two glass microscope slides together, leaving a gap of about 0.5 cm to support aligning eggs and preventing them from rolling over during injections (Figure 3A).</li>
	<li>Apply a piece of transparent film dressing (see Table of Materials) on the double-sided tape in the gap between the slides. Ensure to align the film dressing perfectly with the gap.</li>
</ol>
5. Embryo microinjections
<ol>
	<li>Align 8-10 eggs at a time on the slide setup (above) using a single-haired paintbrush. 
	NOTE: Eggs aligned with the longer axis perpendicular (Figure 3B) to the slide edge are easier to inject than eggs whose longitudinal axis is aligned parallel to the edge (Figure 3C). Eggs in the perpendicular orientation (Figure 3B) can better withstand injection resulting in higher survival.</li>
	<li>Place the slide with aligned eggs on the stage of a compound microscope. Use a piece of lint-free wipe to remove the 1% NaCl solution in which they are stored.</li>
	<li>Attach the filled injection needle to a microinjector connected to a micromanipulator (see Table of Materials).</li>
	<li>Open the needle by gently rubbing it against the egg surface using a high-pressure setting on the microinjector (&#62;5,000 hPa).</li>
	<li>After the needle is opened, reduce the pressure (1,000-2,500 hPa) of the microinjector depending on the opening of the needle. 
	NOTE: A small opening will require comparatively higher pressure, while a larger opening will require a lower pressure. This is to control the volume of the construct injected. Depending on the size of the needle opening, the injection volume will vary from 1-5 pL.</li>
	<li>Inject all the eggs on the slide at a 10&#176;-15&#176; angle as quickly as possible and immediately move the slide to high humidity conditions in a Petri dish lined with moist paper. As soon as the eggs are injected, they start to desiccate. 
	&#8203;NOTE: Injecting a small number of eggs at a time increases the injection success and the survival probability. Needles do get clogged by embryo reflux. If the tip is still sharp, to clear clogged needles, move the needle into a small droplet of 1% NaCl added to the edge of the slide. The capillary action will dislodge the small clog. If the needle is already blunt, change it.</li>
</ol>
6. Post-injection care of embryos
<ol>
	<li>Place the slide containing injected embryos in a large Petri dish lined with moist filter paper. 
	NOTE: It is critical not to disturb the eggs for at least 5-6 h. This allows the injection wound to seal properly. The injection wound may open up when agitated, and cytoplasm may begin to ooze out, resulting in egg mortality.</li>
	<li>After 5-6 h, add a small drop of DI water to the transparent film dressing with injected embryos attached. Using a paintbrush, gently displace the eggs. Transfer the eggs to a small Petri dish (10 cm) and submerge them in DI water. 
	NOTE: The film dressing allows easy removal of eggs after injections. The moment a water drop is added to the film dressing, the eggs will start to move along with the water.</li>
	<li>Keep the eggs submerged in water and place the Petri dish in a 6-quart plastic box in an incubator at 27 &#176;C and &#62;90% RH.</li>
	<li>Check the eggs regularly, daily for the first few days, and then every 3-4 days until larvae start hatching. 
	NOTE: If a large number of eggs are damaged during microinjections, gently draining the DI water after a week will prevent fungal growth on the dead eggs.</li>
	<li>When the larvae begin to hatch from the injected embryos (21-25 days after injection for the present laboratory setup), check them daily and transfer any hatched larvae to glass vials with a screen on top. Larvae can hatch underwater and survive for ~1-2 weeks.</li>
	<li>Screen the larvae4 if the eggs were injected with a construct that may result in a visible phenotype.</li>
</ol>

<ol>
	<li>Hinckley, A. F. et al. Lyme disease testing by large commercial laboratories in the United States. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 59 (5), 676-681 (2014).</li>
	<li>Jongejan, F., Uilenberg, G. The global importance of ticks. Parasitology. 129 Suppl, S3-14 (2004).</li>
	<li>Eisen, R. J., Eisen, L. The blacklegged tick, Ixodes scapularis: An increasing public health concern. Trends in Parasitology. 34 (4), 295-309 (2018).</li>
	<li>Sharma, A. et al. Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control. iScience. 25 (3), 103781 (2022).</li>
	<li>Nuss, A., Sharma, A., Gulia-Nuss, M. Genetic manipulation of ticks: A paradigm shift in tick and tick-borne diseases research. Frontiers in Cellular and Infection Microbiology. 11, 678037 (2021).</li>
	<li>Heinze, S. D. et al. CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus. Scientific Reports. 7 (1), 4582 (2017).</li>
	<li>Kistler, K. E., Vosshall, L. B., Matthews, B. J. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Reports. 11 (1), 51-60 (2015).</li>
	<li>Criscione, F., O&#39;Brochta, D. A., Reid, W. Genetic technologies for disease vectors. Current Opinion in Insect Science. 10, 90-97 (2015).</li>
	<li>Jasinskiene, N. et al. Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proceedings of the National Academy of Sciences of the United States of America. 95 (7), 3743-3747 (1998).</li>
	<li>Dermauw, W. et al. Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae. Insect Biochemistry and Molecular Biology. 120, 103347 (2020).</li>
	<li>Santos, V. T. et al. The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system. Genesis (New York, N.Y. 2000). 51 (12), 803-818 (2013).</li>
	<li>Ginsberg, H. S., Lee, C., Volson, B., Dyer, M. C., Lebrun, R. A. Relationships between maternal engorgement weight and the number, size, and fat content of larval Ixodes scapularis (Acari: Ixodidae). Journal of Medical Entomology. 54 (2), 275-280 (2017).</li>
	<li>Feldman-Muhsam, B., Havivi, Y. Accessory glands of Gene&#39;s organ in ticks. Nature. 187 (4741), 964 (1960).</li>
	<li>Kakuda, H., Koga, T., Mori, T., Shiraishi, S. Ultrastructure of the tubular accessory gland in Haemaphysalis longicornis (Acari: Ixodidae). Journal of Morphology. 221 (1), 65-74 (1994).</li>
	<li>Booth, T. F. Wax lipid secretion and ultrastructural development in the egg-waxing (Gen&#233;&#39;s) organ in ixodid ticks. Tissue &#38; Cell. 21 (1), 113-122 (1989).</li>
	<li>Arrieta, M. C., Leskiw, B. K., Kaufman, W. R. Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae). Experimental &#38; Applied Acarology. 39 (3-4), 297-313 (2006).</li>
	<li>Brady, J. A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bulletin of the World Health Organization. 32 (1), 143-144 (1965).</li>
</ol>

The authors have nothing to disclose.

This is the first protocol developed to inject early tick embryos successfully. A survival rate of ~4%-8% has been achieved, which is comparable to embryo injection in other well-established insect models5.
As this is the initial protocol, it is anticipated that this protocol will be further refined and specialized to individual tick species. In particular, injection timing will vary from species to species, dependent upon embryogenesis, especially the timing of cellula...

Ticks are vectors of medical and veterinary importance, capable of transmitting a variety of viral, bacterial, protozoan pathogens and nematodes1,2. In the eastern United States, the black-legged tick, Ixodes scapularis, is an important vector of the Lyme disease (LD) pathogen, the spirochete Borrelia burgdorferi. Over 400,000 cases of LD are reported each year in the United States, making it the top vector-borne infectious disease in the US1. In addition to B. burgdorferi, six other microorganisms are transmitted by I. scapularis- including four bacteria (Anaplasma phagocytophilum, B. mayonii, B. miyamotoi, and Ehrlichia muris eauclarensis), one protozoan parasite (Babesia microti), and one virus (Powassan virus), making this tick species a major public health concern3. While tick-borne diseases have become more prevalent in recent years, research on ticks has fallen behind other arthropod vectors, such as mosquitoes, due to the unique biology of ticks&#160;and challenges associated with applying genetic and functional genomic tools4,5.
Gene-editing techniques, particularly CRISPR/Cas9, have now made functional genomics studies feasible in non-model organisms. For creating heritable mutations in an organism, embryo injection remains the preferred method for delivering constructs for altering the germline6,7,8,9. However, until recently4, tick eggs were considered too difficult or even impossible to inject without killing the embryo10,11. A thick wax layer on eggs, hard chorion, and high intra-oval pressure were some of the main obstacles that prevented embryo injection in ticks. Adult, blood-fed I. scapularis deposit a single clutch of up to 2,000 eggs12 over 3-4 weeks (approximately 100 eggs/day). Eggs are laid singly, and each egg is coated with wax that is secreted by protrusions or &#34;horns&#34; of the glandular Gen&#233;&#39;s organ13,14,15&#160;of the mother. This wax protects the eggs from desiccation and contains antimicrobial compounds15. To successfully inject tick eggs, it is important to remove the wax layer, soften the chorion, and desiccate the eggs to decrease the intraoval pressure so that the injection does not irreversibly damage the egg. Understanding the critical importance of embryo injections for successful germline transformation, a protocol for I. scapularis is developed, which can be used to deliver a CRISPR/Cas9 construct and generate stable germline mutations4. In addition to its contribution to I. scapularis research, this protocol could also be optimized for other tick species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum silicate capillaries, with filament</td>
			<td>Sutter instruments</td>
			<td>AF100-64-10</td>
			<td>Embryo injection</td>
		</tr>
		<tr>
			<td>Benzalkonium chloride 50% in water, 25 g</td>
			<td>TCI-America</td>
			<td>B0414</td>
			<td>Embryo treatment, 25 g is approximately 25 mL</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001-090</td>
			<td>Post-injection care</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Thomas Scientific</td>
			<td>300-101</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Genesee Scientific</td>
			<td>88-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>Loading the pulled needles</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>ROE-200</td>
			<td>Embryo injection</td>
		</tr>
		<tr>
			<td>Microscopic slides- plain, ground edges</td>
			<td>Genesee Scientific</td>
			<td>29-100</td>
			<td>Embryo alignment, ground edges are preferred, beveled edges could obscure the eggs from view</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Research Products International</td>
			<td>S23020-500.0</td>
			<td>Embryo treatment</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Double sided tape</td>
			<td>Scotch</td>
			<td>34-8716-3417-5</td>
			<td>Embryo alignment</td>
		</tr>
		<tr>
			<td>Petri plates</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td>Post-injection care</td>
		</tr>
		<tr>
			<td>Tegaderm/ Transparent film dressing</td>
			<td>3M Healthcare</td>
			<td>1628</td>
			<td>Embryo alignment</td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td>Fine Science Tools</td>
			<td>10130-10</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>Tungsten Wire</td>
			<td>Amazon</td>
			<td>B08DNT7ZK3</td>
			<td>Gene`s organ manipulation</td>
		</tr>
		<tr>
			<td>XenoWorks Digital Microinjector</td>
			<td>Sutter instruments</td>
			<td>MPC-200</td>
			<td>Embryo injection</td>
		</tr>
	</tbody>
</table>

embryo injection technique for gene editing in the black-legged tick, ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

A successful embryo injection protocol for I. scapularis is described in this article. Egg-laying females were kept at high humidity to avoid desiccation of partially-waxed eggs. The wax layer was removed to inject tick embryos by ablating the Gene&#39;s organ (wax gland) of the gravid female (Figure 1A-E). We used aluminosilicate glass needles with a shorter neck (Figure 2). This shape was ideal for tick egg injection ...

Watch this Scientific Journal Video about Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis at JoVE.com

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

embryo-injection-technique-for-gene-editing-black-legged-tick-ixodes

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2.4K Views.  University of Nevada, Reno. The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Video: Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

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Assay for Neural Induction in the Chick Embryo

Electrophysiological Recording in the Drosophila Embryo

In vitro Reconstitution of the Active T. castaneum Telomerase

Induction and Testing of Hypoxia in Cell Culture

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

In situ Hybridization for Sipunculus nudus Coelomic Fluid

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis