La ricerca qui presentata è stata sostenuta da Charles River. Gli autori sono molto grati ai modelli Genetically Engineered and Services Group e il personale embriologia per la cura degli animali e l&#39;iniezione dell&#39;ormone.

1. Topo crioconservazione degli spermatozoi <ol><li> Per ogni linea da crioconservati, i seguenti elementi prima di iniziare. <ol><li> 1 vasca di un 4-ben piatto con 240 l di kit CPA </li><li> 15 cannucce crioconservazione degli spermatozoi come segue <ol><li> Collegare il 0,25 ml cannucce al siringhe da 1 ml con il tappo di cotone più vicino alla siringa. In ogni paglia, carico di 8 cm (circa 160 microlitri) di media FIV, quindi 1 cm di aria. Etichettare il cannucce con il nome della linea da conservare. </li></ol></li></ol></li><li> LN 2 dovrebbe essere di 15 cm di profondità nella casella di schiuma. Chiudere il coperchio. </li><li> Euthanize 2 topi maschi da ogni riga di essere crioconservati. I topi dovrebbero essere tra 10 e 60 settimane di età. Rimuovere le due epididimi caudale insieme al dotto deferente di ogni topo. </li><li> Posizionare il epididimi nel precedentemente preparato CPA contenenti anche di un 4-ben piatto (passo 1.1). </li><li> Fare 5-7 tagli longitudinali in ogni epididimo e delicatamentespremere ogni dotto deferente per spingere lo sperma fuori. </li><li> Agitare la miscela e CPA epididimo a mano delicatamente a temperatura ambiente per 3-5 minuti. Questo permetterà spermatozoi a nuotare fuori. </li><li> Utilizzando la paglia precedentemente preparato, aspirare 5 mm (circa 10 microlitri) di sospensione di spermatozoi e quindi 1 cm di aria. </li><li> Ripetere questa operazione fino a un totale di 15 cannucce. Carico e di tenuta cannucce entro 10 minuti per la sopravvivenza degli spermatozoi migliori. </li><li> Si chiude entrambi i lati della paglia con attenzione. Posizionare il cannucce nel contenitore con la colonna congelamento dello sperma prima. </li><li> Fissare il 1 pipetta ml fornito come parte del kit per il canestro di estendere la maniglia. Posizionare il contenitore in LN 2 nella casella schiuma attraverso il foro nel coperchio. Lasciare che il canestro di galleggiare verticalmente nel LN 2 per 10 minuti. </li><li> Immergere il contenitore nel LN2. Lo sperma è ora crioconservati e può essere spostato lungo termine LN 2 di stoccaggio. </li><li> Il contenitore non deve essere utilizzato perla riga successiva fino a quando è riscaldato a temperatura ambiente. </li></ol> 2. Topo superovulazione <ol><li> In generale, l&#39;uso di giovani topi di sesso femminile lo stesso sfondo i donatori di sperma. </li><li> I topi devono essere ~ 12 g (3-4 settimane di età). </li><li> Eseguire un&#39;iniezione intraperitoneale con 5-10 UI di gravidanza siero di cavalla gonadotropina (PMSG). </li><li> Attendere 46-48 ore. </li><li> Eseguire un&#39;iniezione intraperitoneale con 5-10 UI di gonadotropina corionica umana (hCG). </li><li> Ovociti vendemmia 14-16 ore dopo l&#39;iniezione di hCG come descritto di seguito. </li></ol> 3. Topo di recupero degli spermatozoi e fecondazione in vitro <ol><li> Prima di fecondazione in vitro, per ogni paglia di essere scongelati, i seguenti elementi ed equilibrare per almeno un&#39;ora a 37 ° C CO 2 incubatore. <ol><li> Spermatozoi piatto di incubazione: un piatto 35 millimetri Petri, con un calo 90 l di media sperma trattamento (STM) ricoperto di olio. </li><li> Taglio piatto: Per la raccolta degli ovociti da un massimo di 10 superovulated femmine, una 35 millimetri Petri piatto con 3 ml di terreno fecondazione in vitro. </li><li> FIV bene: per ogni gruppo di 5 femmine superovulated per la FIV, un pozzetto di un piatto ben 4 con 500 l di media FIV (se si utilizza superovulated 10 femmine, per esempio, avrete bisogno di 2 pozzi...) </li><li> Lavaggio delle stoviglie: una piatto 35 millimetri Petri con 3 ml di simplex di potassio medio ottimizzato (KSOM) per lavare ogni pozzetto del 4-ben piatto FIV utilizzate </li><li> Cultura piatto: un piatto di 35 millimetri di Petri con 50 gocce l di media KSOM ricoperto di olio per coltura degli embrioni da ogni pozzetto del piatto FIV. </li></ol></li><li> Rimuovere una cannuccia da LN 2 e mettere in un bagno d&#39;acqua a 37 ° C per 2-3 minuti. </li><li> Rimuovere la paglia dal bagnomaria e pulire per rimuovere l&#39;acqua in eccesso. Effettuare il primo taglio vicino alla colonna d&#39;aria al più vicino la spina cotone. Tagliare la paglia nel medio FIV ci saranno ancora una colonna d&#39;aria visibile prima della colonna sperma. </li><li> Poi tagliare la fine della paglia nel seconda colonna d&#39;aria, facendo attenzione a evitare di tagliare la colonna sperma. Tagliata in un angolo di 45 - questo renderà molto più facile per aspirare lo sperma. </li><li> Aspirare la sospensione dello sperma a partire dalla fine distale della paglia con una pipetta 200 l. </li><li> Posizionare la sospensione spermatozoi nel centro della goccia STM e incubare il piatto per 45-60 minuti a 37 ° C 5% CO 2 incubatore. </li><li> Durante l&#39;incubazione degli spermatozoi, sezionare gli ovidotti delle femmine superovulated nel piatto di taglio. Utilizzare un ago 30 g o pinze per strappare ogni ovidotto, rilasciando il cumulo-ovocita complessi (COC). Evitare grassi e sangue nel medio FIV come sporco dei media rende fasi di lavaggio in più. </li><li> Una volta che tutti gli ovidotti sono stati strappati, trasferire i contraccettivi orali combinati da 5 femmine per ogni pozzi fecondazione in vitro. Il numero di COC prodotta da superovulazione è molto sforzo dipendente. </li><li> Raccogliere 10-15 ml di sperma dalla periferia della goccia STM e fertilizzare uno FIV e circa 14-16 ore dopo hCG injezione. Ripetere per ogni fecondazione ben utilizzato. Concentrazione dello sperma finale sarà di circa 1 milione / ml. </li><li> Co-coltura spermatozoi e ovociti per 4-6 ore a 37 ° C 5% CO 2 incubatore. </li><li> Lavare gli embrioni con KSOM un minimo di 2 volte e la cultura in gocce KSOM per lo sviluppo a 37 ° C 5% CO 2 incubatore. </li><li> Tasso di fecondazione in vitro è stato calcolato come embrioni di cellule a doppio stadio di ovociti totale utilizzata dopo una notte della cultura. </li></ol> 4. Rappresentante Risultati Spermatozoi dal 5 Charles River ceppi di topi inbred è stato congelato. Rapporti di protezione sono calcolati dividendo gli spermatozoi congelati i valori di valutazione da valori freschi valutazione degli spermatozoi. Rapporti di protezione della motilità degli spermatozoi sono stati 60,2%, 81,3%, 78,6%, 66,5%, 61,0% per C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, rispettivamente. Rapporti di protezione per una rapida motilità progressiva sono stati 47,3%, 69,4%, 57,0%, 52,8%, 54,1% in C57BL/6NCrl, 129S2/SvPas<html> Crl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, rispettivamente (Fig. 1). I tassi di fecondazione in vitro con gli spermatozoi congelati-scongelati sono stati il ​​55,7%, 26,4%, 87,3%, 87,8% e 26,2% nel C57BL/6NCrl, 129S2/SvPasCrl, FVB / NCrl, DBA/2NCrl, e BALB / cAnNCrl, rispettivamente (Fig. 2) . Nel corso di un periodo di 1,5 anni, 49 linee di topo geneticamente modificato sono stati archiviati dalla crioconservazione degli spermatozoi. Queste linee sono stati successivamente recuperati con successo (come definito da cuccioli nati vivi) da FIV in 4 ceppi di sesso femminile (C57BL / 6, BALB / c, DBA / 1 e 129Sv) (Fig. 3). <img alt="Figura 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> Figura 1. I rapporti Tutela della motilità spermatica e la motilità progressiva rapida nei topi <img alt="Figura 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> Figura 2. Fecondazione in vitro con lo sperma congelato, scongelato in topi 3713fig3.jpg &quot;/&gt; Figura 3. Fecondazione in vitro con spermatozoi congelati-scongelati in topi 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>

Gli autori sono dipendenti del Charles River, il creatore di questo kit.

Dal 1990, la base del protocollo di congelamento degli spermatozoi con il 18% raffinosio e il 3% di latte scremato è stato dimostrato affidabile in molti ceppi di topi e un gran numero di linee del mouse sono stati crioconservati 1,2,3,4,5,6,7. Le principali cause di danni alle cellule dello sperma durante il processo di congelamento e scongelamento sono stati associati con la formazione di ghiaccio 8,9 e le specie reattive dell&#39;ossigeno 10. La supplementazione con pulitori del radicale libero, come gli amminoacidi, e agenti riducenti, come monotioglicerolo, nel mezzo di congelamento è stato studiato e dimostrato di aumentare notevolmente tasso di fecondazione in vitro con lo sperma congelato, scongelato in ceppi inbred compreso il C57BL / 6 11,6. Nel presente protocollo, abbiamo sviluppato un nuovo I • Cryo kit per la crioconservazione degli spermatozoi del mouse attraverso l&#39;ottimizzazione di base disponibili in commercio CPA con antiossidanti e inibitori del ghiaccio e modificando il congelamento degli spermatozoi e la procedura di fecondazione in vitro. Rapporti di protezione della motilità degli spermatozoi(&gt; 60%) e la motilità progressiva rapida (&gt; 45%) sono state osservate per gli spermatozoi provenienti da cinque ceppi di topi inbred congelati con la Cryo • I kit. Il 2-cellula embrionale tasso fase di sviluppo con lo sperma congelato, scongelato in 5 ceppi di topi inbred è stato notevolmente migliorato rispetto a quello con base 11,6 CPA. Utilizzando il kit, l&#39;utente crea una sospensione concentrata di spermatozoi dello sperma di congelamento di 2 maschi per ogni linea. Solo da congelamento 15 cannucce il processo è veloce, facile e occupa meno spazio di memorizzazione per l&#39;archiviazione a breve oa lungo termine. La maggior parte delle volte, le linee del mouse può essere recuperato da scongelamento solo 1 o 2 cannucce.

<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

Migliaia di nuovi geneticamente modificati (GM) ceppi di topi sono stati creati dopo l&#39;avvento di tecnologie di transgenesi e di eliminazione diretta. Molti di questi animali preziosi esistono solo come gli animali vivi, senza alcun piano di backup in caso di emergenza. Crioconservazione degli embrioni in grado di fornire il backup, ma è costoso, può essere una procedura lunga e richiede generalmente un gran numero di animali per il successo. Dopo la scoperta che gli spermatozoi del mouse può essere crioconservati con successo con un agente di base crioprotettivi (CPA), composto da 18% raffinosio e il 3% di latte scremato, la crioconservazione degli spermatozoi è diventata una procedura accettabile e conveniente per l&#39;archiviazione, la distribuzione e il recupero di questi ceppi di valore . Qui mostriamo un nuovo sviluppo • I kit per la crioconservazione degli spermatozoi Cryo mouse. Sperma di cinque ceppi di uso comune di topi inbred sono stati congelati con questo kit e poi recuperato. Rapporti di protezione più elevato della motilità degli spermatozoi (&gt;60%) e la motilità progressiva rapida (&gt; 45%) rispetto al controllo (di base CPA) sono stati osservati per lo sperma congelato con questo kit di 5 ceppi di topi inbred. Due stadi di sviluppo embrionale delle cellule dopo la fecondazione in vitro con lo sperma recuperato è stato migliorato costantemente in tutte e 5 ceppi di topi esaminati. Nel corso di un periodo di 1,5 anni, 49 linee di topo geneticamente modificato sono stati archiviati dalla crioconservazione degli spermatozoi con la Cryo • I kit e successivamente recuperato da fecondazione in vitro.

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

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

Qui mostriamo il nuovo • I kit per la crioconservazione degli spermatozoi Cryo mouse. Stadio di due cellule sviluppo dell&#39;embrione con decongelati sperma era migliorato costantemente in 5 ceppi di topi con l&#39;utilizzo di questo kit. Nel corso di un periodo di 1,5 anni, 49 linee di topi geneticamente modificati sono stati archiviati dalla crioconservazione degli spermatozoi con la Cryo • I kit e poi recuperato con successo da fecondazione in vitro.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

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

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Biology

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

39.3K Views.  Charles River. Qui mostriamo il nuovo • I kit per la crioconservazione degli spermatozoi Cryo mouse. Stadio di due cellule sviluppo dell'embrione con decongelati sperma era migliorato costantemente in 5 ceppi di topi con l'utilizzo di questo kit. Nel corso di un periodo di 1,5 anni, 49 linee di topi geneticamente modificati sono stati archiviati dalla crioconservazione degli spermatozoi con la Cryo • I kit e poi recuperato con successo da fecondazione in vitro.

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

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.

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il 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 ...

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Biologia

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.

Immunoistochimica: sezioni di paraffina Utilizzando il Vectastain ABC Kit Labs Vector

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

Estrazione RNA da cellule Neuroprecursor Utilizzando il Bio-Rad RNA totale 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.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

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.

Genotipizzazione rapida del tessuto mouse utilizzando Sigma Extract-N-Amp PCR Tissue 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.

Raccolta e Crioconservazione degli ovociti e di embrioni di topo criceto

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.

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

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.

Crioconservazione di embrioni di topo da glicole etilenico-Based Vetrificazione

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

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.

Valutazione di sperma di pesce utilizzando Software e dispositivi di raffreddamento

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

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Raccolta di sperma di qualità differenziale mediante centrifugazione in gradiente di densità

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Trasformazione naturale, espressione proteica e crioconservazione del cianobatterio filamentoso Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...