A pesquisa aqui apresentada foi apoiada por Charles River. Os autores são muito gratos ao Modelos Geneticamente Modificadas e grupo de serviços e os funcionários Embriologia para cuidar dos animais e injeção de hormônio.

1. Criopreservação de esperma do mouse <ol><li> Para cada linha a ser criopreservados, prepare o seguinte antes de começar. <ol><li> Um bem de um prato 4-bem com 240 l de kit CPA </li><li> 15 de esperma palhas de criopreservação da seguinte forma <ol><li> Conecte a 0,25 ml palhas para a 1 ml seringas com o tampão de algodão mais próximo à seringa. Em cada palha, carga de 8 cm (aproximadamente 160 L) de meio de fertilização in vitro, em seguida, um centímetro de ar. Rotular os canudos com o nome da linha a ser preservado. </li></ol></li></ol></li><li> LN 2 deve ser de 15 cm de profundidade na caixa de espuma. Feche a tampa. </li><li> Euthanize dois camundongos machos de cada linha a ser criopreservados. Ratos deve estar entre 10 e 60 semanas de idade. Remova os dois epidídimos caudal juntamente com o canal deferente de cada rato. </li><li> Coloque o epidídimos no CPA previamente preparado contendo além de um prato 4-bem (passo 1.1). </li><li> 07/05 fazer cortes longitudinais em cada epidídimo e gentilmenteespremer cada vaso deferente para empurrar os espermatozóides para fora. </li><li> Agitar a mistura de CPA e epidídimo com a mão suavemente em temperatura ambiente por 3-5 minutos. Isto permitirá que os espermatozóides de nadar para fora. </li><li> Usando a palha preparada anteriormente, aspirado 5 mm (aproximadamente 10 ml) de suspensão de esperma e depois de 1 cm de ar. </li><li> Repita esse processo com até um total de 15 canudos. De carga e lacre canudos dentro de 10 minutos para a sobrevivência melhor esperma. </li><li> Sele ambos os lados da palha com cuidado. Coloque as palhetas na caixinha de congelamento com a coluna de esperma em primeiro lugar. </li><li> Anexar a pipeta de 1 ml fornecidos como parte do kit para a caixinha para estender a alça. Coloque o recipiente em LN 2 na caixa de espuma através do furo na tampa. Permitir que o recipiente para flutuar verticalmente na LN 2 para 10 minutos. </li><li> Mergulhe o canister no LN2. O esperma é agora criopreservados e pode ser movido para longo prazo LN 2 de armazenamento. </li><li> O recipiente não deve ser usado paraa próxima linha até que ele é aquecido à temperatura ambiente. </li></ol> 2. Superovulação do mouse <ol><li> Geralmente, o uso camundongos fêmeas jovens da mesma origem que os doadores de esperma. </li><li> Ratos deve ser ~ 12 g (3-4 semanas de idade). </li><li> Realizar uma injeção intraperitoneal com UI de Pregnant mare 10/05 gonadotrofina sérica (PMSG). </li><li> Aguarde 46-48 horas. </li><li> Realizar uma injeção intraperitoneal com UI 10/05 da gonadotrofina coriônica humana (hCG). </li><li> Oócitos colheita h após a injeção de hCG 14-16, conforme descrito abaixo. </li></ol> 3. Recuperação do mouse esperma e fertilização in vitro <ol><li> Antes de fertilização in vitro, para cada palha para ser descongeladas, prepare o seguinte e estabilizar pelo menos uma hora em 37 ° C incubadora de CO 2. <ol><li> Prato de esperma de incubação: uma placa de Petri 35 milímetros, com uma queda de 90 mL de meio de esperma tratamento (STM) coberto com óleo. </li><li> Corte prato: Para a coleta de oócitos de um máximo de 10 superovulated fêmeas, uma 35 milímetros placa de Petri com 3 ml de meio de fertilização in vitro. </li><li> IVF bem: Para cada grupo de 5 fêmeas superovuladas para fertilização in vitro, um bem de um prato 4 bem com 500 mL de meio de fertilização in vitro (se você estiver usando 10 fêmeas superovuladas, por exemplo, você vai precisar de 2 poços...) </li><li> De lavar loiça: uma 35 milímetros placa de Petri com 3 ml de meio de potássio simplex otimizado (KSOM) para lavar cada poço da placa IVF-4 bem utilizado </li><li> Placa de cultura: um 35 milímetros placa de Petri com 50 mL de meio de gotas KSOM coberto com azeite, para cultura de embriões a partir de cada poço da placa IVF. </li></ol></li><li> Remover uma palha de LN 2 e coloque em banho-maria a 37 ° C por 2-3 minutos. </li><li> Remover a palha do banho de água e limpe a remover o excesso de água. Fazer o primeiro corte perto da coluna de ar mais próxima primeiro o tampão de algodão. Cortar a palha no meio de fertilização in vitro ainda haverá uma coluna de ar visíveis antes da coluna de esperma. </li><li> Em seguida, corte a ponta do canudo na sEGUNDA coluna de ar, tendo o cuidado de evitar o corte da coluna de esperma. Cortada num ângulo de 45 - isto irá tornar muito mais fácil para aspirar o espermatozóide. </li><li> Aspirar a suspensão de espermatozóides a partir da extremidade distal da palha com uma pipeta de 200 mL. </li><li> Lugar a suspensão de espermatozóides no centro da gota STM e incubar o prato por 45-60 minutos em 37 ° C de 5% CO 2 incubadora. </li><li> Durante a incubação de espermatozóides, dissecar os ovidutos das fêmeas superovuladas no prato de corte. Use uma agulha de 30 g ou uma pinça para arrancar cada oviduto, liberando os complexos cumulus-oócito (COCs). Evite gordura e sangue no meio de fertilização in vitro como sujos mídia faz para as etapas de lavagem extra. </li><li> Depois que todos os ovidutos foram rasgadas, transferir o COCs de 5 mulheres para cada poços de FIV. O número de COCs produzidos por superovulação é muito tensão dependente. </li><li> Coletar 10-15 mL de esperma a partir da periferia da queda STM e fertilizar uma fertilização in vitro bem aproximadamente 14-16 horas pós-inj hCGç ã o. Repita a fertilização para cada bem utilizados. Concentração espermática final será aproximadamente 1 milhão / ml. </li><li> Co-cultura de esperma e ovócitos para 4-6 horas a 37 ° C de 5% incubadora de CO 2. </li><li> Lavar os embriões com KSOM um mínimo de 2 vezes e cultura em gotas KSOM para o desenvolvimento de um 37 ° C de 5% incubadora de CO 2. </li><li> Taxa de fertilização in vitro foi calculada como dois embriões de células estágio de oócitos total utilizada após cultura durante a noite. </li></ol> 4. Resultados representante Esperma de cinco Charles cepas puras Rio rato foi congelado. Relações de proteção são calculadas dividindo-se os valores de avaliação do esperma congelado por valores nova avaliação do esperma. Razões de protecção da mobilidade do esperma foram 60,2%, 81,3%, 78,6%, 66,5%, 61,0% para C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, respectivamente. Relações de proteção para a motilidade progressiva rápida foram 47,3%, 69,4%, 57,0%, 52,8%, 54,1% em C57BL/6NCrl, 129S2/SvPas<html> Crl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, respectivamente (Fig. 1). As taxas de fertilização in vitro com os espermatozóides congelados e descongelados foram 55,7%, 26,4%, 87,3%, 87,8% e 26,2% em C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, respectivamente (Fig. 2) . Durante um período de 1,5 anos, 49 linhas do mouse GM foram arquivados por criopreservação de esperma. Estas linhas foram mais tarde recuperados com sucesso (como definido pela filhotes nascidos vivos) por FIV em 4 cepas do sexo feminino (C57BL / 6, BALB / c, DBA / 1 e 129Sv) (Fig. 3). <img alt="Figura 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> Figura 1. Rácios de Proteção da motilidade espermática e motilidade progressiva rápida em Ratos <img alt="Figura 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> Fertilização Figura 2. In Vitro com esperma congelado-descongelado em Ratos 3713fig3.jpg &quot;/&gt; Figura 3. Fertilização in vitro com esperma congelado-descongelado em camundongos GM

<ol>
	<li>Tada, N., Sato, M., Yamanoi, J., Mizorogi, T., Kasai, K., Ogawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+in+the+presence+of+raffinose+and+glycerol.">Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol.</a> J. Reprod. Fertil. 89, 511-516 (1990).</li><li>Yokoyama, M., Akiba, H., Katsuki, M., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+normal+young+following+transfer+of+mouse+embryos+obtained+by+in+vitro+fertilization+using+cryopreserved+spermatozoa.">Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa.</a> Jikken Dobutsu. 39, 125-128 (1990).</li><li>Sztein, J. M., Farley, J. S., Young, A. F., Mobraaten, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+cryopreserved+mouse+spermatozoa+affected+by+temperature+of+collection+and+rate+of+thawing.">Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.</a> Cryobiology. 35, 46-52 (1997).</li><li>Thornton, C. E., Brown, S. D., Glenister, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+numbers+of+mice+established+by+in+vitro+fertilization+with+cryopreserved+spermatozoa:+implications+and+applications+for+genetic+resource+banks,+mutagenesis+screens,+and+mouse+backcrosses.">Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.</a> Mamm. Genome. 10, 987-992 (1999).</li><li>Sztein, J. M., Farley, J. S., Mobraaten, L. E. <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+fertilization+with+cryopreserved+inbred+mouse+sperm.">In vitro fertilization with cryopreserved inbred mouse sperm.</a> Biol. Reprod. 63, 1774-1780 (2000).</li><li>Liu, L., Nutter, L. M., Law, N., McKerlie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+freezing+and+in+vitro+fertilization+in+three+substrains+of+C57BL/6+mice.">Sperm freezing and in vitro fertilization in three substrains of C57BL/6 mice.</a> J. Am. Assoc. Lab. Anim. Sci. 48, 39-43 (2009).</li><li>Nakagata, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+mouse+spermatozoa+and+in+vitro+fertilization.">Cryopreservation of mouse spermatozoa and in vitro fertilization.</a> Methods. Mol. Biol. 693, 57-73 (2011).</li><li>Mazur, P., Koshimoto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+intracellular+ice+formation+the+cause+of+death+of+mouse+sperm+frozen+at+high+cooling+rates.">Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates.</a> Biol. Reprod. 66, 1485-1490 (2002).</li><li>Jin, B., Yamasaki, C., Yamada, N., Seki, S., Valdez, D. M., Kasai, M., Edashige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+mouse+spermatozoa+are+injured+during+freezing.">The mechanism by which mouse spermatozoa are injured during freezing.</a> J. Reprod. Dev. 54, 265-269 (2008).</li><li>Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., Diekman, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+signaling+pathways+involved+in+sperm+acquisition+of+fertilizing+capacity.">Novel signaling pathways involved in sperm acquisition of fertilizing capacity.</a> J. Reprod. Immunol. 53, 133-150 (2002).</li><li>Ostermeier, G. C., Wiles, M. V., Farley, J. S., Taft, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserving,+distributing+and+managing+genetically+modified+mouse+lines+by+sperm+cryopreservation.">Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation.</a> PLoS ONE. 3, e2792-e2792 (2008).</li></ol>

Os autores são funcionários da Charles River, o fabricante deste kit.

Desde 1990, o protocolo de congelamento de esperma básica usando rafinose 18% e 3% de leite desnatado provou ser confiável em muitas cepas de camundongos e um grande número de linhas do mouse foram criopreservados 1,2,3,4,5,6,7. As principais causas de danos às células de esperma durante o congelamento e descongelamento processo têm sido associados com a formação de gelo 8,9 e espécies reativas de oxigênio 10. Suplementação com varredores de radicais livres, tais como aminoácidos e agentes redutores, tais como monotioglicerol, no meio de congelamento foi investigada e comprovada para aumentar substancialmente a taxa de fertilização in vitro com os espermatozóides congelados e descongelados em linhagens puras, incluindo a C57BL / 6 11,6. No protocolo atual, que desenvolveu um novo I • Cryo kit de criopreservação de esperma do mouse através da otimização do CPA básica com antioxidantes comercialmente disponíveis e bloqueadores de gelo e modificando o congelamento de espermatozóides e procedimento de fertilização in vitro. Razões de protecção da motilidade espermática(&gt; 60%) e motilidade progressiva rápida (&gt; 45%) foram observados para o esperma de cinco linhagens de camundongos inbred congelados com a Cryo • Eu kit. O 2-célula estágio taxa de desenvolvimento do embrião com esperma congelado-descongelado em 5 linhagens de camundongos inbred foi marcadamente melhorado em comparação ao que, com base CPA 11,6. Ao utilizar o kit, o usuário cria uma suspensão de espermatozóides concentrado por congelamento de esperma 2 machos para cada linha. Por apenas 15 canudos de congelamento do processo é rápido, fácil e ocupa menos espaço de armazenamento para o armazenamento de curto ou longo prazo. Na maioria das vezes, as linhas do mouse podem ser recuperados por descongelamento apenas 1 ou 2 canudos.

<table border="1">
	<tbody>
		<tr>
			<th>
				Materials</th>
			<th>
				Supplier</th>
			<th>
				Catalogue number</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>CPA</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>STM</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>IVF medium</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>0.25 ml plastic straw</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Sperm freezing canister</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Cane and goblet</td>
			<td>Charles River</td>
			<td>&nbsp;</td>
			<td>Provided with kit</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5310</td>
			<td>Should be embryo tested</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-106-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>Falcon</td>
			<td>351008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat sealer</td>
			<td>ABTEC</td>
			<td>TISH-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-well multidish</td>
			<td>Nunc</td>
			<td>73521-424</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Gilson</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C + 5% CO2 incubator</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LN2 storage system</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37&deg;C water bath</td>
			<td>VWR</td>
			<td>89032-196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>Intervet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>EMDChemicals</td>
			<td>367222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1cc syringe w/ 26G 3/8 needle</td>
			<td>BD</td>
			<td>BD309625</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 gauge &frac12; needle</td>
			<td>BD</td>
			<td>305106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-5135, RS-5110, RS-5005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

mouse sperm cryopreservation and recovery using the i&middot;cryo kit

Milhares de novos geneticamente modificados (GM) linhagens de camundongos foram criados desde o advento das tecnologias de transgênese e knockout. Muitos desses animais valiosos existem apenas como animais vivos, sem plano de backup em caso de emergência. Criopreservação de embriões pode fornecer esta cópia de segurança, mas é caro, pode ser um processo lento, e geralmente requer um grande número de animais para o sucesso. Desde a descoberta que o esperma do mouse podem ser criopreservados com sucesso com um agente básico crioprotectores (CPA), constituído por rafinose 18% e 3% de leite desnatado, a criopreservação de esperma tornou-se um procedimento aceitável e de baixo custo para arquivamento, distribuição e recuperação dessas cepas valiosa . Aqui demonstramos um recém-desenvolvido I • kit Cryo para mouse criopreservação de esperma. Esperma de cinco cepas comumente usados ​​de camundongos isogênicos foram congelados utilizando este kit e depois se recuperou. Maior proteção dos rácios de motilidade espermática (&gt;60%) e motilidade progressiva rápida (&gt; 45%) em relação ao controle (básico CPA) foram vistos por esperma congelado com este kit em 5 linhagens de camundongos inbred. Duas células desenvolvimento embrionário palco após a fertilização in vitro com o esperma recuperado foi melhorado de forma consistente em todas as cinco linhagens de camundongos examinados. Durante um período de 1,5 anos, 49 linhas do mouse GM foram arquivados por criopreservação de espermatozóides com a Cryo kit • I e mais tarde recuperado por fertilização in vitro.

Watch this Scientific Journal Video about Mouse Sperm Cryopreservation and Recovery using the IÃ‚Â·Cryo Kit at JoVE.com

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Aqui demonstramos a recém-desenvolvida I • kit Cryo para mouse criopreservação de esperma. Duas células desenvolvimento embrionário palco com descongelado esperma foi melhorado consistentemente em cinco linhagens de camundongos com a utilização deste kit. Durante um período de 1,5 anos, 49 linhas de camundongos geneticamente modificados foram arquivados por criopreservação de espermatozóides com a Cryo kit • I e mais tarde recuperados com sucesso por fertilização in vitro.

Criopreservação do mouse Sperm and Recovery utilizando o I · Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

mouse-sperm-cryopreservation-and-recovery-using-the-icryo-kit

Research

JoVE Journal

Biology

39.5K visualizações.  Charles River. Aqui demonstramos a recém-desenvolvida I • kit Cryo para mouse criopreservação de esperma. Duas células desenvolvimento embrionário palco com descongelado esperma foi melhorado consistentemente em cinco linhagens de camundongos com a utilização deste kit. Durante um período de 1,5 anos, 49 linhas de camundongos geneticamente modificados foram arquivados por criopreservação de espermatozóides com a Cryo kit • I e mais tarde recuperados com sucesso por fertil...

Vídeo: Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Imuno-histoquímica: Seções de parafina Usando o Kit de Vectastain ABC Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Extração de RNA a partir de células Neuroprecursor Usando o Bio-Rad Total de RNA Kit

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

A genotipagem rápida de tecido Mouse Usando Extract-N-Amp Tissue Sigma PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Recolha e criopreservação de óvulos e embriões de camundongos Hamster

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Criopreservação de embriões de camundongos por Vitrificação Etileno Glicol-Based

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Avaliação do esperma de peixe utilizando Software e dispositivos de refrigeração

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...