Name: a capsule-based model for immature hard tick stages infestation on laboratory mice
Uploaded: 2020-07-09T14:00:00
Description: Watch this Scientific Journal Video about A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice at JoVE.com

We acknowledge the technical assistance of Alain Bernier French National Institute of Agricultural Research (INRAE), and Oc&#233;ane Le Bidel (ANSES). The study was supported by the DIM One Health - R&#233;gion &#206;le-de-France (Acronym of the project: NeuroPaTick). The mice were purchased by ANSES. Dr. Jeffrey L. Blair is acknowledged for reviewing the earlier version of the manuscript.

Please note that this protocol can be only applied when all welfare and safety measures are met in the laboratory. This protocol received permission to use mice for tick feeding by the Ethics Committee for Animal ExperimentsComEth Anses/ENVA/UPEC, Permit Numbers E 94 046 08. For the endpoint, the animals were exposed to CO2 for 9 min in two phases of 4 and 5 min each one.
1. Preparation of the capsule
<ol>
	<li>Stick 2 mm thick EVA-foam and the adhesive double sticky foam together...

<ol>
	<li>Sonenshine, D. E., Roe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Ticks. , (2014).</li><li>Kr&#246;ber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends in Parasitology. 23 (9), 445-449 (2007).</li><li>Bonnet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transstadial+and+transovarial+persistence+of+Babesia+divergens+DNA+in+Ixodes+ricinus+ticks+fed+on+infected+blood+in+a+new+skin-feeding+technique.">Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique.</a> Parasitology. 134 (2), 197-207 (2007).</li><li>Bonnet, S., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+artificial+infection+of+hard+ticks:+A+tool+for+the+analysis+of+tick-borne+pathogen+transmission.">Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission.</a> Acarologia. 52 (4), 453-464 (2012).</li><li>Kohls, G. M., Galtsoff, P. S., Lutz, F. E., Welch, P. S., Needham, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+rearing+methods+with+special+reference+to+the+Rocky+Mountain+wood+tick,+Dermacentor+andersoni.">Tick rearing methods with special reference to the Rocky Mountain wood tick, Dermacentor andersoni.</a> Culture methods for invertebrate animals. , 246-256 (1937).</li><li>Faccini, J. L. H., Chacon, S. C., Labruna, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbits+(Oryctolagus+cuniculus)+as+experimental+hosts+for+Amblyomma+dubitatum.+Neumann+(Acari:+Ixodidae).">Rabbits (Oryctolagus cuniculus) as experimental hosts for Amblyomma dubitatum. Neumann (Acari: Ixodidae).</a> Arquivo Brasileiro de Medicina Veterin&#225;ria e Zootecnia. 58 (6), 1236-1239 (2006).</li><li>Chacon, S. 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E., Maramorsch, K., Mahmood, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+ticks+in+the+laboratory.">Maintenance of ticks in the laboratory.</a> Maintenance of Human, Animal, and Plant Pathogen Vectors. , 57-82 (1999).</li><li>Levin, M. L., Schumacher, L. B. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+different+isolates+of+Anaplasma+phagocytophilum+by+Ixodes+scapularis+from+a+model+animal.">Acquisition of different isolates of Anaplasma phagocytophilum by Ixodes scapularis from a model animal.</a> Vector Borne Zoonotic Diseases. 4 (1), 53-59 (2004).</li><li>Heinze, D. M., Wikel, S. K., Thangamani, S., Alarcon-Chaidez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+murine+cutaneous+response+during+initial+and+subsequent+infestations+with+Ixodes+scapularis+nymphs.">Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis nymphs.</a> Parasites &amp; Vectors. 6 (5), 26 (2012).</li><li>Nuss, A. B., Mathew, M. G., Gulia-Nuss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing,+Ixodes+scapularis,+the+Black-legged+Tick:+Feeding+Immature+Stages+on+Mice.">Rearing, Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice.</a> Journal of Visualized Experiments. (123), e55286 (2017).</li><li>Wada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+ablation+of+basophils+in+mice+reveals+their+nonredundant+role+in+acquired+immunity+against+ticks.">Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.</a> Journal of Clinical Investigation. 120 (8), 2867-2875 (2010).</li><li>Saito, T. B., Walker, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+feeding+by+the+Rocky+Mountain+spotted+fever+vector,+Dermacentor+andersoni,+induces+interleukin-4+expression+by+cognate+antigen+responding+CD4++T+cells.">Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells.</a> Parasites &amp; Vectors. 2 (1), 47 (2009).</li><li>Gargili, A., Thangamani, S., Bente, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+laboratory+animal+hosts+on+the+life+cycle+of+Hyalomma+marginatum+and+implications+for+an+in+vivo+transmission+model+for+Crimean-Congo+hemorrhagic+fever+virus.">Influence of laboratory animal hosts on the life cycle of Hyalomma marginatum and implications for an in vivo transmission model for Crimean-Congo hemorrhagic fever virus.</a> Frontiers in Cell and Infection Microbiology. 20 (3), 39 (2013).</li><li>Almaz&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Versatile+Model+of+Hard+Tick+Infestation+on+Laboratory+Rabbits.">A Versatile Model of Hard Tick Infestation on Laboratory Rabbits.</a> Journal of Visualized Experiments. (140), e57994 (2018).</li><li>Zhijun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+and+biological+characteristics+of+Dermacentor+silvarum+Olenev+(Acari:+Ixodidae)+under+field+conditions.">The life cycle and biological characteristics of Dermacentor silvarum Olenev (Acari: Ixodidae) under field conditions.</a> Veterinary Parasitology. 168 (3-4), 323-328 (2010).</li><li>Ahmed, B. M., Taha, K. M., El Hussein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Hyalomma+anatolicum+Koch,+1844+(Acari:+Ixodidae)+fed+on+rabbits,+sheep+and+goats.">Life cycle of Hyalomma anatolicum Koch, 1844 (Acari: Ixodidae) fed on rabbits, sheep and goats.</a> Veterinary Parasitology. 177 (3-4), 353-358 (2011).</li><li>&#352;irok&#253;, P., Erhart, J., Petr&#382;elkov&#225;, K. J., Kamler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+tortoise+tick+Hyalomma+aegyptium+under+laboratory+conditions.">Life cycle of tortoise tick Hyalomma aegyptium under laboratory conditions.</a> Experimental and Applied Acarology. 54, 277-284 (2011).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Haemaphysalis+doenitzi+(Acari:+Ixodidae)+under+laboratory+conditions+and+its+phylogeny+based+on+mitochondrial+16S+rDNA.">Life cycle of Haemaphysalis doenitzi (Acari: Ixodidae) under laboratory conditions and its phylogeny based on mitochondrial 16S rDNA.</a> Experimental and Applied Acarology. 56, 143-150 (2012).</li><li>Jin, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+Cycle+of+Dermacentor+everestianus+Hirst,+1926+(Acari:+Ixodidae)+under+Laboratory+Conditions.">Life Cycle of Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae) under Laboratory Conditions.</a> Korean Journal of Parasitology. 55 (2), 193-196 (2017).</li><li>Labruna, M. B., Fugisaki, E. Y., Pinter, A., Duarte, J. M., Szab&#243;, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+and+host+specificity+of+Amblyomma+triste+(Acari:+Ixodidae)+under+laboratory+conditions.">Life cycle and host specificity of Amblyomma triste (Acari: Ixodidae) under laboratory conditions.</a> Experimental and Applied Acarology. 30 (4), 305-316 (2003).</li><li>Breuner, N. 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The most critical step in the protocol is firm gluing of the capsule to the mouse skin. Therefore, the latex glue should be homogenously applied to the entire EVA-foam surface of the capsule and constant pressure for 3 minutes should be applied, especially to the left and right side of the capsule. We also recommend placement of the capsule as far forward on the back as possible to avoid its removal by the mouse using its rear paws. In our experiments, only the adhesion of the EVA-foam and latex glue to the mouse skin ha...

Ticks are important vectors of several pathogens and represent a serious risk to animal and human health1. Setting up an effective feeding system is crucial when studying their biology, tick-host-pathogen interactions, or establishing effective control measures. Currently, several artificial feeding systems, which avoid the use of live animals are available for ticks2,3,4 and these should be utilized whenever experimental conditions allow. However, in various experimental settings these systems fail to appropriately mimic the specific physiologic features and the use of live animals is necessary to achieve relevant results.
Laboratory mice are commonly used for the study of many biological systems and are routinely utilized as hosts for feeding ticks5,6,7,8,9. The two most common methods of feeding immature ticks on mice include free infestations and the use of confinement chambers attached to the mouse. Free infestations are primarily used for larval stages and engorged ticks can drop to an area where they can be recovered. Confinement chambers are usually composed of acrylic or polypropylene caps which are glued to the mouse&#8217;s back. The first technique is an effective natural system for tick feeding but does not allow close monitoring during the experiment because the individual ticks are dispersed in different parts of the host body. Additionally, engorged ticks that drop to a recovery area can become contaminated with feces and urine10,11,12,13,14 that may severely affect the tick fitness or they can be damaged or eaten by the mouse if there is no separation between the animal and the recovery area15. Chamber-based systems allow the confinement of ticks to a defined area, however, the gluing process is laborious and the caps are often weakly adherent to the glue and thus they often detach during the experiment16,17,18,19. The caps are also stiff, uncomfortable, and lead to skin reactions, which prevent the re-use of the mice and necessitates their euthanasia after the experiment.
In our previous study, we successfully developed an effective system using chambers made of ethylene-vinyl acetate (EVA) foam for feeding ticks on laboratory rabbits20. Herein, we adapted this system to a mouse model and propose a simple and clean method to feed immature hard tick stages in closed capsules made from EVA-foam. Specifically, our system uses elastic EVA-foam capsules glued to the shaved mice back with fast drying (3 min), non-irritating latex glue. This technique allows firm and long-lasting attachment of capsules to the experimental mouse, as well as effective tick infestation/collection during the entire course of the experiment. The flat capsule is made from flexible materials and does not impede manipulation of the mouse for blood collection or other purposes. The system is suitable mainly for the nymphal tick stages, but with slight modification it can be used for feeding larvae as well. The method can be completed by one single experienced person and extensive training is not required.

<table>
	<tbody>
		<tr>
			<td>EVA-foam 2 mm thick, (low density)</td>
			<td>Cosplay Shop</td>
			<td>EVA-45kg (950/450/2 mm)</td>
			<td>It can be ordered also via Amazon (ref. no. B07BLMJDXD)</td>
		</tr>
		<tr>
			<td>Heat Shrink Tubing Electric Wire Wrap Sleeve</td>
			<td>Amazon</td>
			<td>B014GMT1AM</td>
			<td>Different diameters of Heat Shrink Tubing are available via Amazon.</td>
		</tr>
		<tr>
			<td>Mice BALB/cByJ</td>
			<td>Charles River</td>
			<td>Strain code 627</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice C57BL/6</td>
			<td>Charles River</td>
			<td>Strain code 664</td>
			<td></td>
		</tr>
		<tr>
			<td>No-toxic Latex Glue</td>
			<td>Tear mender</td>
			<td>Fabric &#38; Leather Adhesive</td>
			<td>Also available also via Amazon (ref. no. B001RQCTUU)</td>
		</tr>
		<tr>
			<td>Punch Tool Hand Art Tool</td>
			<td>Amazon</td>
			<td>B07QPWNGBF</td>
			<td>Saled by amazon as Leather Working Tools 1-25mm Round Steel Leather Craft Cutter Working for Belt Strap</td>
		</tr>
		<tr>
			<td>PVC Binding Covers Transparent</td>
			<td>Amazon</td>
			<td>B078BNLSNP</td>
			<td>Any transparent PVC sheet of ticknes between 0.150 mm to 0.180 mm is suitable</td>
		</tr>
		<tr>
			<td>Self Adhesive Pad Sponge Double Coated Foam Tape</td>
			<td>Amazon</td>
			<td>B07RHDZ35J</td>
			<td>Saled by amazon as 2 Rolls Double Sided Foam Tape, Super Strong White Mounting Tape Foam</td>
		</tr>
		<tr>
			<td>Transparent seal stickers (20 mm diameter circles)</td>
			<td>Amazon</td>
			<td>B01DAA6X66</td>
			<td></td>
		</tr>
	</tbody>
</table>

a capsule-based model for immature hard tick stages infestation on laboratory mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

We propose the detailed step-by step method for feeding immature hard tick stages in EVA-foam capsules applied to a mouse&#8217;s back (Figure 2). This non-laborious protocol is suitable for various types of experiments when precise tick monitoring and collection is required. The main advantages of this method are its simplicity, easily accessible cost-effective materials, and short duration. In addition, we succeeded in attaching two capsules to one mouse individual (Fi...

Watch this Scientific Journal Video about A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice at JoVE.com

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

Our technique is suitable for various types of experiments but our mouse model is required for feeding ticks and is necessary to keep the ticks in enclosed area for easy collection and monitoring. The benefit of this simple method is it efficiency, short duration, and the ability to monitor or collect ticks at different time points of an experiment. Our system is suitable for studying tick biology, host-vector pathogen interactions or evaluating different control measure of these medically important arthropods.This method requires accuracy and attention to detail as immature tick stages are small and feeding them on mice necessitates the enclosed capsule to be be firmly attached to the mouse for several days. Begin by sticking together a two millimeter EVA foam, an adhesive double sticky foam. Use a 20 millimeter diameter leather hole punch to cut a circle from the foam pieces.Then, use a 12 millimeter hole punch to cut the interior. Peel the protective paper strip from the adhesive double sticky foam and attach a 20 millimeter transparent plastic circle to the foam. If feeding larvae, do not remove the protective paper strip from the adhesive foam and proceed directly with capsule attaching.Make a one centimeter slit in the transparent plastic then use an entomological pin to create at least 10 small holes for moisture evaporation during the experiment. After the mouse has been anesthetized place it on the manipulation pad and attach a nose cone for continuous isoflurane supply. Ensure that the breathing rate is less than 80 breaths per minute by adjusting the isoflurane level.Shave the anterior part of the mouse from behind the shoulder blades up to the area just behind the ears creating a shaved area that is greater than the capsule surface. Apply non irritating latex glue to the entire EVA foam site of the capsule and wait for one minute. Place the capsule on the mouse's back and apply constant pressure for three minutes especially on the left and right side of the capsule.Slightly lift the capsule to visually check its attachment to the skin. To ensure the firm attachment of the capsule apply a slight three minute constant pressure with the fingers especially on the left and right side of the capsule. If non-attached regions are found, apply more glue with a spatula and press for another three minutes.If a non-attachment region forms during the experiment fix it in the same way. Introduce individual nymphs into the capsule via the one centimeter slit. Slightly squeeze the capsule from two sides to allow the transparent plastic to bend then push the nymph inside the capsule.Turn the forceps 90 degrees and pull them out. For larvae infestation, remove the paper slip from the attached capsule and place the syringe with the larvae directly inside. Deposit the ticks by pushing the syringe plunger then gently turn the plunger towards the skin to remove the remaining attached larvae.Once the larvae are deposited onto the skin close the capsule by attaching the transparent plastic circle. Cut a 3.5 millimeter strip of the protective plastic band and apply the protective plastic band around the capsule then return the mouse to the cage. To collect the ticks, create a cross shaped cut in the plastic with a scalpel and use forceps to collect the ticks.If necessary, reclose the capsule by sticking an adhesive plastic patch on the transparent plastic. the same plastic patch can be used if collection of ticks at multiple time points is required. Alternatively, the mouse can be euthanized and the ticks collected or manually detached after capsule removal.For successful recovery of the mice, keep them in a cage for one additional week and let the capsules detach naturally. Once the capsule is off, check for abnormal reactions on the skin. Apply any emollient lotion if there is any irritation.This protocol makes it possible to feed immature hard ticks in EVA foam capsules applied to a mouse's back. The use of the fast drying and non irritating latex glue ensures that the capsule is firmly attached within three minutes and remains attached for at least seven days. Attaching two capsules makes it possible to feed two different groups of ticks on the same animal.Furthermore, complete recovery of the mice after the experiments, makes it possible to reuse the animals and avoid euthanasia. This system has been successfully used to feed Ixodes ricinus nymphs. A moderate to high engorgement success rate was achieved in two different mouse strains.In both strains, all nymphs finished the feeding within four to five days while the majority dropped off on the fourth day. When attempting this protocol, it's important to shave a sufficient area of mouse's skin and effectively attach the capsule to avoid it's accidental detachment during the experiment. Besides the study of basic tick biology, this method can be used to explore pathogen transmission from tick to host or host to ticks.Additionally, the option to reuse the mouse offers a possibility for multiple re-infestations that can be beneficial for immunological studies or investigating different tick-borne pathogen transmissions.

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Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

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.

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.

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