Agradecemos Jose Noguera assistência precoce com massagem cloacal, e Jeffrey Kittilson para laboratório de apoio. Este trabalho foi apoiado por um prêmio de ND EPSCoR FAR0022429 a BJH. Custos de publicação foram generosamente apoiados pelo departamento de ciências biológicas da NDSU.

Este protocolo envolve assuntos animais vertebrados e foi aprovado pelo Comitê de uso (IACUC) da North Dakota State University e institucional Cuidado Animal.
1. coleta de sêmen de uma ave passeriforme da família usando esfregaços massagem
Nota: Esfregaços massagem é uma técnica de coleção de sêmen eficaz sobre aves reprodutivamente ativas somente, mas pode ser realizada com sucesso fora da época de reprodução em situações apropriadas em cativeiro. Atividade reprodutiva deve ser determinada para a espécie-alvo antes de utilizar a massagem esfregaços. Esta técnica pode ser usada em aves selvagens após captura ou em pássaros mantidos em cativeiro.
<ol><li>Começando com um pássaro masculino, (em condição de reprodução) aderência do bander na mão não dominante, transferir o pássaro para a mão dominante com seu lado ventral, tocando a palma (Figura 1).<ol>
<li>Certifique-se de que a cabeça da ave é o mais próximo a borda lateral da palma da mão. Seguro levemente a cabeça e o corpo usando o mindinho, anel e os dedos do meio. Isso deixa do pássaro respiradouro e cauda exposta.</li>
		<li>Deixe as pernas do pássaro solto ou levemente comedido, usando a palma da mão.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> Figura 1 : Aperto para massagem esfregaços. O pássaro está seguro levemente na mão dominante, com seu lado ventral, tocando a palma e cabeça garantidas pelo dedo mindinho. <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"><li>Coloque o dominante o dedo indicador e o polegar de cada lado da extremidade superior da cloaca e levemente belisca os dedos juntos, aplicando uma pressão muito leve em direção a cauda ( Figura 2). A cloaca será evert, expondo a mucosa cloacal (interior cor de rosa). Nota: Se o pássaro não está atualmente produzindo sêmen, a protuberância cloacal pode ser pequena e a cloaca é difícil de evert. Com a experiência, é fácil diferenciar entre as cloacas de machos reprodutivamente ativas e inativas5,11.</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> Figura 2 : Dedo posição e protuberância cloacal. Evert cloaca aplicando pressão ligeira à extremidade superior (A). Em um ativo reprodutivamente masculino ave passeriforme da família, a protuberância cloacal é óbvia e empresa (B). <a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<ol start="3"><li>Com a mão não dominante, coloque a almofada do dedo no lado dorsal da base da cauda para estabilizar a mão e em seguida, coloque a ponta do polegar logo abaixo da cloaca de sempre. Nota: Uma miniatura curta e Lisa é vantajosa para a realização de massagem cloacal e protege o pássaro de lesão acidental.</li>
	<li>Mova o polegar não-dominante cranialmente e profunda em um movimento repetitivo com média pressão, ficando abaixo da cloaca. Continue até o pássaro ejacula. Dependendo do indivíduo, isto normalmente leva entre 1 e várias dezenas de cursos.</li>
	<li>Assim que o pássaro ejacula, colete o sêmen em um tubo de Microhematócrito. Repita a massagem esfregaços para obter múltiplos ejacula. Nota: Passeriformes sémen é castanho claro e normalmente tem uma consistência um pouco grossa. Substâncias brancas ou escuras, expelidas da cloaca não estão sémen e podem contaminar a amostra de sêmen. A aparência de uma pequena quantidade de sangue na cloaca ou misturado com o sêmen é geralmente causado por muita pressão durante a massagem.</li>
	<li>Para a extração de DNA, coloca o sêmen em um tubo de microcentrifugadora com 20 µ l de 1X PBS (pH 7,4). Nota: Sêmen armazenado em PBS 1x não é apropriado para a inseminação artificial, mas pode ser usado para a extração de DNA em uma data posterior quando armazenado a-80 ° C. O PBS pode ser temperatura ambiente ou refrigerados, mas deve estar no estado líquido, quando a amostra de sêmen é depositada.<ol>
<li>Confirme a presença de espermatozoides na amostra exibindo uma pequena quantidade da amostra diluída sob um microscópio composto (Figura 3).</li>
	</ol>
</li>
	<li>Armazene o sêmen diluído a-80 ° C até a extração.</li>
</ol>2. extração de DNA do sêmen do pássaro
Nota: Este protocolo foi testado com vários jogos comerciais de extração de DNA, mas só faz sucesso quando usado com um DNA kit12. Etapas que são modificadas desde o protocolo do kit são indicadas por *.
<ol><li>Preparar uma solução 1m de ditiotreitol (DTT) em água e misture usando um vortex.*<ol>
<li>Alíquota da solução de TDT e armazená-lo a-20 ° C até imediatamente antes da utilização. Cada amostra a ser extraído requer 10 µ l de solução de TDT * 1m</li>
	</ol>
</li>
	<li>Preparar um buffer contendo 20 mM Tris-Cl, pH 8.0; 20 mM EDTA; 200 mM NaCL; e 4% SDS. Cada amostra a ser extraído exige 90 µ l desta buffer.*<ol>
<li>Armazene o buffer em temperatura ambiente. Se ele se precipitou, aquecê-lo a 56 ° C, para dissolve.*</li>
	</ol>
</li>
	<li>Pré-aqueça uma incubadora contendo um balancim ou agitador a 65 ° c</li>
	<li>Imediatamente antes de iniciar a extração, misture a solução de TDT 10 µ l com 90 µ l de buffer preparado no passo 2.2 para cada amostra a ser extraído, por exemplo, 900 buffer de µ l além de 100 µ l TDT solução se extraindo 10 amostras. Esta mistura será chamada TDT-buffer.*</li>
	<li>Adicionar 70 µ l 1X PBS para a amostra de sêmen diluído inteiro e misture bem, usando um vortex.*</li>
	<li>Adicione 100 µ l tampão-DTT e solução de proteinase K 10 µ l de amostra. Vórtex por 20 segundos, então incubar na incubadora pré-aquecido por 1 hora em um balancim ou shaker.*</li>
	<li>Adicionar 200 µ l de buffer AL e 200 µ l EtOH fresco (100%) e vórtice para encerrar 20</li>
	<li>Transferir a amostra inteira para uma coluna de rotação e centrifugar 1 minuto em 6.000 x. g. descarte o tubo de coleta.</li>
	<li>Coloque a coluna em um tubo novo e juntar 500 µ l preparado tampão AW1. Centrifugar 1 minuto em 6.000 x. g. descarte o tubo de coleta.</li>
	<li>Coloque a coluna em um tubo novo e juntar 500 µ l preparado tampão AW2. Centrífuga para 1 minuto em 6.000 g. x descartar o escoamento e coloque a coluna de volta no tubo.</li>
	<li>Centrifugar a 3 minutos a 20.000 x g. descarte o tubo de coleta e colocar a coluna em um tubo de microcentrifugadora de 1,5 mL.</li>
	<li>Pipeta 35 µ l de AE buffer directamente sobre a membrana da coluna e incubar a temperatura ambiente por 5 minutes.*</li>
	<li>Centrifugar 1 minuto a 14.000 x. g. descartar a coluna de rotação e armazenar amostras extraídas a-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>

Os autores declaram que eles têm não tem interesses financeiro concorrente.

Nós descrevemos um método simples e confiável para coleta de sêmen de aves pequenas e médias em condição reprodutivamente ativa e extrair DNA de esperma aviária.
O protocolo de extração de DNA de sémen descrito é modificado a partir de um kit para manter a simplicidade, mas foi refinado para uso em sêmen aviária. Espermatozoides são resistentes à Lise por extração padrão produtos químicos9,13, e amostras de sêmen c...

Pássaros são temas ideais para estudos que envolvem a qualidade do esperma e competição1, esperma telômero dinâmica2, epigenetics3e tópicos semelhantes, como são amplamente utilizados em pesquisas biológicas e esperma pode ser degustadas facilmente usando esfregaços massagem. Esfregaços massagem é uma técnica manual suave, não-invasivo para a coleta de sêmen de aves4,5,6. Amostras repetidas podem ser obtidas facilmente e sem ferramentas especiais são necessárias, tornando simples para executar no campo ou no laboratório. Apesar de esfregaços massagem tem sido usado por décadas, é difícil de aprender com os escritos descrições disponíveis. Esta publicação destina-se a reduzir o tempo e incerteza envolvidos em aprender massagem esfregaços.
Sêmen coletado de aves usando esfregaços massagem pode ser usado imediatamente para avaliação de motilidade4,7 ou inseminação artificial8, ou congelado para outros usos, tais como de imagem avançada e extração de DNA. Amostras de sêmen de aves da ordem Passeriformes são pequenas mas contenham esperma densamente. DNA é extraído usando um kit de extração comercial para manter a simplicidade, com modificações para superar as funções de protecção especializadas de esperma9. Após a extração, telômeros de esperma podem ser medidos usando 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

Colheita de sémen pode ser útil para uma vasta gama de aplicações, incluindo estudos que envolvem a qualidade do esperma, esperma telômero dinâmica e epigenética. Pássaros são assuntos amplamente utilizados na investigação biológica e são ideais para estudos que envolvam amostras repetidas de esperma. No entanto, alguns recursos estão atualmente disponíveis para aqueles que desejam aprender a coletar e extrair DNA de esperma aviária. Aqui descrevemos esfregaços massagem, uma técnica manual suave, não-invasivo para a coleta de esperma aviária. Embora esta técnica é estabelecida na literatura, pode ser difícil de aprender com as descrições disponíveis. Nós também fornecemos informações para extrair DNA de sémen aviária usando um kit de extração comercial com modificações. Esfregaços massagem pode ser usada facilmente em qualquer pequeno para médio porte masculino ave condição reprodutiva. Após a coleta, o sêmen pode ser usado imediatamente para ensaios de motilidade ou congelado para extração de DNA, seguindo o protocolo descrito neste documento. Este protocolo de extração foi refinado por esperma aviária e tem sido utilizado com sucesso em amostras coletadas de diversas espécies de passeriformes (Passer domesticus, Spizella passerina, Haemorhous mexicanuse Turdus migratorius ) e um columbid (Columba livia).

Massagem de esfregaços e DNA extração usando que o protocolo descrito foi realizado em várias espécies de passeriformes e um columbid (tabela 1). A presença de espermatozoides em amostras de sêmen recolhidas pela massagem esfregaços foi confirmada pela visualização de uma pequena quantidade das amostras diluídas sob um microscópio composto em ampliação de 400 X (Figura 3). Depois de extrair o DNA de amostras de sêmen coletados ...

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

Este protocolo descreve esfregaços massagem, uma técnica não-invasiva manual para coleta de sêmen de aves e extração de DNA de esperma aviária usando um kit de extração comercial com modificações.

Coleta de sêmen aviária por esfregaços massagem e isolamento do DNA de esperma

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

avian-semen-collection-cloacal-massage-isolation-dna-from

Research

JoVE Journal

Biology

15.9K visualizações.  North Dakota State University. Este protocolo descreve esfregaços massagem, uma técnica não-invasiva manual para coleta de sêmen de aves e extração de DNA de esperma aviária usando um kit de extração comercial com modificações.

Vídeo: 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.

Sperm colheita e Inseminação Artificial de Ratos

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 ...

Biologia

Isolation of Genomic DNA from Mouse Tails

Isolamento de DNA genômico de Tails rato

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.

Isolamento Islet humana Pancreatic: Parte I: Digestão e Coleta de tecido pancreático

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.

Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

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.

Isolamento e In vitro A ativação do 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 ...

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.

Um método prático e inovador para extrair DNA genômico de Kits de coleta de sangue para Preservação 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.

Isolamento, coleta e Enriquecimento de bactérias naturais magnetotáticas do Meio Ambiente

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.

Transferência de Embrião Utero-tubária e vasectomia no Rato Modelo

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.

A cultura<em&gt; Caenorhabditis elegans</em &gt; Em Axenic Liquid Media e Criação de transgênicos Worms por bombardeamento com micropartículas

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.

DNA Eletroporação, Isolamento e imagem das miofibras

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