Nous remercions aide précoce avec massage cloacal, Jose Noguera et Jeffrey Kittilson pour laboratoire appuyer. Ce travail a été soutenu par un prix de ND EPSCoR FAR0022429 à BJH. Frais de publication ont été généreusement financés par le département des Sciences biologiques de NDSU.

Ce protocole comporte des sujets animaux vertébrés et a été approuvé par l’animalier institutionnel et utilisation Comité (IACUC) à North Dakota State University.
1. prélèvement d’une espèce d’oiseau à l’aide de massage cloacale
NOTE : Cloacale massage est une technique de collection de sperme efficace seulement des oiseaux actifs sexuellement, mais peut être réalisée avec succès à l’extérieur de la saison de reproduction dans les situations appropriées en captivité. Activité reproductrice doit être déterminée pour les espèces cibles avant d’utiliser le massage cloacale. Cette technique peut servir à la capture des oiseaux sauvages ou sur les oiseaux gardés en captivité.
<ol><li>Commençant par un oiseau mâle, (en état de se reproduire) poignée de bander dans la main non dominante, transférer l’oiseau dans la main dominante avec sa face ventrale, toucher la paume (Figure 1).<ol>
<li>Assurez-vous que la tête de l’oiseau est plus proche du bord latéral de la paume. Fixer légèrement la tête et le corps à l’aide de le pinky, anneau et le médius. Cela laisse de l’oiseau vent et queue exposés.</li>
		<li>Laisser les pattes de l’oiseau libre ou légèrement bloqués à l’aide de la paume.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Figure 1 : Grip pour massage cloacale. L’oiseau est attaché légèrement dans la main dominante avec sa face ventrale, toucher la paume et la tête garanti par le petit doigt. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">S’il vous plaît cliquez ici pour visionner une version agrandie de cette figure.</a>
<ol start="2"><li>Placez l’index et pouce dominante de chaque côté de l’extrémité supérieure du cloaque et légèrement pincer les doigts ensemble tout en appliquant une pression très légère vers la queue ( Figure 2). Le cloaque sera evert, exposant la muqueuse cloacale (rose intérieur). Remarque : Si l’oiseau n’est pas en train de produire le sperme, la protubérance cloacale peut être petite et le cloaque est difficile à evert. Avec l’expérience, il est facile de faire la différence entre les cloaques des mâles sexuellement actifs et inactifs5,11.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Figure 2 : Finger position et protubérance cloacale. Leonie le cloaque en appliquant une légère pression à l’extrémité supérieure (A). Dans un passereau mâle actif sexuellement, la protubérance cloacale est évidente et entreprise (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">S’il vous plaît cliquez ici pour visionner une version agrandie de cette figure.</a>
<ol start="3"><li>Avec la main non dominante, placez le tampon de l’index sur la face dorsale de la base de la queue pour stabiliser la main et ensuite placer le bout du pouce juste en dessous du cloaque éversé. Remarque : Une miniature courte et lisse est avantageuse pour l’exécution de massage cloacale et protège l’oiseau des blessures accidentelles.</li>
	<li>Déplacer le pouce non dominantes profonde et proche d’un mouvement répétitif avec moyenne pression, restant sous le cloaque. Continuez jusqu'à ce que l’oiseau éjacule. Selon les individus, cela prend en général entre 1 et plusieurs courses de dizaines.</li>
	<li>Dès que l’oiseau éjacule, recueillir le sperme dans un tube de microhematocrit. Répétez cloacale massage pour obtenir les éjaculats multiples. NOTE : Passereaux sperme est brun clair et a généralement une consistance un peu épaisse. Blancs ou noirs des substances s’est éjectés le cloaque ne sont pas sperme et susceptibles de contaminer l’échantillon de sperme. L’apparition d’une petite quantité de sang sur le cloaque ou mixtes en avec le sperme est habituellement causé par une pression trop grande pendant le massage.</li>
	<li>Pour l’extraction de l’ADN, déposer le sperme dans un tube de microcentrifuge 20 µl de solution de 1 PBS x (pH 7,4). NOTE : Sperme stocké dans du PBS 1 x n’est pas adapté à l’insémination artificielle, mais peut être utilisé pour l’extraction d’ADN à une date ultérieure lorsqu’il est conservé à-80 ° C. Le PBS peut être à température ambiante ou réfrigérée, mais devrait être à l’état liquide lorsque l’échantillon de sperme est déposé.<ol>
<li>Confirmer la présence de spermatozoïdes dans l’échantillon en affichant une petite quantité de l’échantillon dilué au microscope composé (Figure 3).</li>
	</ol>
</li>
	<li>Conserver le sperme dilué à-80 ° C jusqu'à l’extraction.</li>
</ol>2. extraction de l’ADN du sperme de l’oiseau
Remarque : Ce protocole a été testé avec plusieurs kits commerciaux d’extraction d’ADN, mais il a seulement réussi lorsqu’il est utilisé avec un ADN kit12. Étapes qui sont modifiés depuis le protocole de la trousse sont indiqués par *.
<ol><li>Préparer une solution de 1 M de dithiothréitol (DTT) dans l’eau et mélanger à l’aide d’un vortex.*<ol>
<li>Partie aliquote de la solution DTT et conserver à-20 ° C jusqu'à ce que juste avant l’emploi. Chaque échantillon à extraire nécessite 10 µL de 1 M TNT TomTom.*</li>
	</ol>
</li>
	<li>Préparer un tampon contenant 20 mM Tris-Cl, pH 8,0 ; 20 mM EDTA ; 200 mM NaCL ; et 4 % SDS. Chaque échantillon à extraire requiert 90 µL de cette buffer.*<ol>
<li>Stocker le tampon à la température ambiante. Si elle a précipité, réchauffer à 56 ° C à dissolve.*</li>
	</ol>
</li>
	<li>Préchauffer un incubateur contenant une bascule ou un shaker à 65 ° C.*</li>
	<li>Immédiatement avant de commencer l’extraction, mélanger 10 µL de solution de TNT avec 90 µL du tampon préparé à l’étape 2.2 pour chaque échantillon à extraire, de p. ex. 900 µL de tampon de plus de 100 µL de solution de TNT s’extraire 10 échantillons. Ce mélange s’appellera DTT-buffer.*</li>
	<li>Ajouter 70 µL 1 x PBS à l’échantillon de sperme dilué entière et mélanger bien à l’aide d’un vortex.*</li>
	<li>Ajouter 100 µL tampon de DTT et 10 µL de solution de protéinase K à l’échantillon. Vortexer pendant 20 secondes, puis incuber dans l’incubateur préchauffé pendant 1 heure sur une bascule ou shaker.*</li>
	<li>Ajouter 200 µL tampon AL et 200 µL EtOH fraîche (100 %) et vortex pour 20 secondes.*</li>
	<li>Transférer la totalité de l’échantillon à une colonne de spin et centrifuger 1 minute à 6 000 x g. jeter le tube de prélèvement.</li>
	<li>Placer la colonne sur un nouveau tube et ajouter 500 µL préparé tampon AW1. Centrifuger 1 minute à 6 000 x g. jeter le tube de prélèvement.</li>
	<li>Placer la colonne sur un nouveau tube et ajouter 500 µL préparé tampon AW2. Centrifuger pendant 1 minute à 6 000 x g. jeter les intermédiaires et les replacer la colonne sur le tube.</li>
	<li>Centrifuger à 3 minutes à 20 000 x g. jeter le tube de prélèvement et placer la colonne sur un tube de microtubes de 1,5 mL.</li>
	<li>Pipette 35 µL de AE tampon directement sur la membrane de la colonne et incuber à température ambiante pendant 5 minutes</li>
	<li>Centrifuger 1 minute à 14 000 x g. jeter la colonne et stocker les échantillons extraits à-80 ° C.</li>
</ol>

<ol>
	<li>Briskie, J. V., Montgomerie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+size+and+sperm+competition+in+birds.">Sperm size and sperm competition in birds.</a> Proc. R. Soc. B. 247, 89-95 (1992).</li><li>Monaghan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+and+life+histories:+the+long+and+the+short+of+it.">Telomeres and life histories: the long and the short of it.</a> Ann. N.Y. Acad. Sci. 1206, 130-142 (2010).</li><li>Carrell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+the+male+gamete.">Epigenetics of the male gamete.</a> Fertility and Sterility. 97 (2), 267-274 (2012).</li><li>Burrows, W. H., Quinn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collection+of+spermatozoa+from+the+domestic+fowl+and+turkey.">The collection of spermatozoa from the domestic fowl and turkey.</a> Poultry Science. 16 (1), 19-24 (1937).</li><li>Wolfson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cloacal+protuberance:+a+means+for+determining+breeding+condition+in+live+male+birds.">The cloacal protuberance: a means for determining breeding condition in live male birds.</a> Bird-Banding. 23 (4), 159-165 (1952).</li><li>Kast, T. L., Ketterson, E. D., Nolan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+ejaculate+quality+in+dark-eyed+juncos+according+to+season,+stage+of+reproduction,+and+testosterone+treatment.">Variation in ejaculate quality in dark-eyed juncos according to season, stage of reproduction, and testosterone treatment.</a> The Auk. 115 (3), 684-693 (1998).</li><li>Helfenstein, F., Podevin, M., Richner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+morphology,+swimming+velocity,+and+longevity+in+the+house+sparrow+Passer+domesticus.">Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus.</a> Behav. Ecol. Sociobiol. 64, 557-565 (2010).</li><li>Purchase, C. F., Earle, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modifications+to+the+IMAGEJ+computer+assisted+sperm+analysis+plugin+greatly+improve+efficiency+and+fundamentally+alter+the+scope+of+attainable+data.">Modifications to the IMAGEJ computer assisted sperm analysis plugin greatly improve efficiency and fundamentally alter the scope of attainable data.</a> J. Appl. Icthyol. 28, 1013-1016 (2012).</li><li>Gill, P., Jeffreys, A. J., Werrett, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forensic+application+of+DNA+'fingerprints'.">Forensic application of DNA 'fingerprints'.</a> Nature. 318 (12), 577-579 (1985).</li><li>Criscuolo, F., 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=Real-time+quantitative+PCR+assay+for+measurement+of+avian+telomeres.">Real-time quantitative PCR assay for measurement of avian telomeres.</a> J. Avian Biol. 40, 342-347 (2009).</li><li>Lovette, I. J., Fitzpatrick, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QIAamp DNA Micro Handbook. , (2014).</li><li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs.">A differential DNA extraction method for sperm on the perivitelline membrane of avian eggs.</a> Mol. Ecol. 9, 2149-2154 (2000).</li><li>Birkhead, T. R., Buchanan, K. L., Devoogd, T. J., Pellatt, E. J., Catchpole, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Song,+sperm+quality+and+testes+asymmetry+in+the+sedge+warbler.">Song, sperm quality and testes asymmetry in the sedge warbler.</a> Anim. Behav. 53, 965-971 (1997).</li><li>L&#252;pold, S., Calhim, S., Immler, S., Birkhead, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+morphology+and+sperm+velocity+in+passerine+birds.">Sperm morphology and sperm velocity in passerine birds.</a> Proc. R. Soc. B. 276, 1175-1181 (2009).</li><li>Samour, J. H., Smith, C. A., Moore, H. D., Markham, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semen+collection+and+spermatozoa+characteristics+in+budgerigars+(Melopsittacus+undulates).">Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulates).</a> Vet. Rec. 118, 397-399 (1986).</li><li>Stelzer, G., Crosta, L., B&#252;rkle, M., Krautwalt-Junghanns, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attempted+semen+collection+using+the+massage+technique+and+semen+analysis+in+various+psittacine+species.">Attempted semen collection using the massage technique and semen analysis in various psittacine species.</a> J. Avian Med. Surg. 19 (1), 7-13 (2005).</li></ol>

Les auteurs déclarent qu’ils n’ont aucun intérêt financier concurrentes.

Nous décrivons une méthode simple et fiable de collecte de sperme de petits et moyens oiseaux en état actif sexuellement et d’extraire l’ADN des spermatozoïdes aviaire.
Le protocole d’extraction d’ADN sperme décrit est modifié par rapport à un kit par souci de simplicité, mais a été affiné pour une utilisation sur sperme aviaire. Spermatozoïdes résistent à la lyse par extraction standard produits chimiques9,13, et...

Les oiseaux sont des sujets idéaux pour les études concernant la qualité du sperme et compétition1, sperme télomère dynamique2, épigénétique3et sujets similaires, car ils sont largement utilisés dans la recherche biologique et le sperme peut être dégustée facilement à l’aide de cloacale massage. Cloacale massage est une technique manuelle douce, non invasive pour recueillir le sperme des oiseaux4,5,6. Échantillons répétés peuvent être obtenus facilement et sans outils spéciaux sont nécessaires, rendant simple à effectuer dans le domaine ou le laboratoire. Bien que le massage cloacale a été utilisé pendant des décennies, il est difficile d’apprendre de l’écrit des descriptions disponibles. Cette publication est destinée à réduire le temps et l’incertitude impliqués dans l’apprentissage de massage cloacale.
Sperme collecté des oiseaux à l’aide de massage cloacale peut être utilisé immédiatement pour la motilité évaluation4,7 ou insémination artificielle8, ou congelé pour d’autres usages tels que l’imagerie avancée et l’extraction d’ADN. Des échantillons de sperme de passereaux sont petites mais contiennent le sperme dense. L’ADN est extrait en utilisant un kit d’extraction commerciale pour plus de simplicité, avec les adaptations pour surmonter les dispositifs de sécurité spécialisées de sperme9. Après extraction, télomères de sperme peuvent être mesurées à l’aide de qPCR10.

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microhematocrit capillary tubes</td>
			<td style="width:71px;">Fisherbrand</td>
			<td style="width:119px;">22-362566</td>
			<td style="width:167px;">Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tubes</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1x PBS</td>
			<td style="width:71px;">Any</td>
			<td style="width:119px;"></td>
			<td>Used for storing semen samples prior to DNA extraction.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DL-Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">10708976001</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1233508</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793566</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SDS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1614363</td>
			<td>Any supplier can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA micro kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">56304</td>
			<td>Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us.&#160;</td>
		</tr>
	</tbody>
</table>

avian semen collection by cloacal massage and isolation of dna from sperm

Collecte de sperme peut être utile pour un large éventail d’applications, y compris les études portant sur la qualité du sperme, sperme télomère dynamique et épigénétique. Les oiseaux sont des sujets largement utilisés dans la recherche biologique et sont idéales pour les études portant sur des échantillons de sperme répétées. Cependant, peu de ressources est actuellement disponibles pour ceux qui souhaitent apprendre à collecter et à extraire l’ADN des spermatozoïdes aviaire. Nous décrivons ici cloacale massage, une technique manuelle douce, non invasive pour la collecte de sperme aviaire. Bien que cette technique est établie dans la littérature, il peut être difficile d’apprendre à partir des descriptions disponibles. Nous fournissons également des informations pour extraire l’ADN de sperme aviaire à l’aide d’un kit d’extraction commerciale sous réserve de modifications. Massage cloacale peut être utilisé facilement sur n’importe quel oiseau mâle petites à moyennes en état de se reproduire. Après la collecte, le sperme peut être utilisé immédiatement pour des dosages de motilité, ou congelé pour l’extraction d’ADN suivant le protocole décrit ci-après. Ce protocole d’extraction a été affinée pour les spermatozoïdes aviaire et a été utilisé avec succès sur des échantillons prélevés dans plusieurs espèces de passereaux (Passer domesticus, Spizella passerina, Haemorhous mexicanuset Turdus migratorius ) et un columbid (Columba livia).

Massage cloacale et ADN extraction en utilisant que le protocole décrit ont été effectué sur plusieurs espèces de passereaux et un columbid (tableau 1). La présence de spermatozoïdes dans le sperme prélèvements cloacaux massage a été confirmée en affichant une petite quantité des échantillons dilués sous un microscope composé au grossissement X 400 (Figure 3). Après que l’extraction d’ADN provenant d’échantillons de sp...

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Ce protocole décrit cloacale massage, une technique non-invasive manuelle de collecte de sperme sur les oiseaux et l’extraction d’ADN de sperme aviaire à l’aide d’un kit d’extraction commerciale sous réserve de modifications.

Collecte de sperme aviaire Massage cloacale et l’isolement de l’ADN des spermatozoïdes par

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

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Biology

15.8K vues.  North Dakota State University. Ce protocole décrit cloacale massage, une technique non-invasive manuelle de collecte de sperme sur les oiseaux et l’extraction d’ADN de sperme aviaire à l’aide d’un kit d’extraction commerciale sous réserve de modifications.

Vidéo: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Le sperme de récolte et l&#39;insémination artificielle des souris

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

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Isolement de l&#39;ADN génomique de souris Tails

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Isolement îlots pancréatiques humaines: Partie I: La digestion et la perception des tissus pancréatiques

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolement et culture de cellules progénitrices embryonnaires aviaires valvulaire

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Une méthode pratique et roman d&#39;extraire l&#39;ADN génomique à partir de kits de prélèvement sanguin pour la préservation Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Collecte, l&#39;isolement et l&#39;enrichissement des bactéries présentes naturellement magnétotactique de l&#39;Environnement

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Utéro-tubaire transfert d&#39;embryons et la vasectomie dans le modèle de souris

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Culture<em&gt; Caenorhabditis elegans</em &gt; En Axenic Liquid Media et création d&#39;transgéniques Worms par bombardement de microparticules

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

ADN électroporation, isolement et d&#39;imagerie de myofibres

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Collecte de sperme aviaire Massage cloacale et l’isolement de l’ADN des spermatozoïdes par

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