Burada sunulan araştırma, Charles River tarafından desteklenmiştir. Yazarlar, hayvan bakımı ve hormon enjeksiyonu için Genetiği Modeller ve Hizmetler grubu ve Embriyoloji personeli için çok teşekkür ediyoruz.

1. Fare sperm dondurulması <ol><li> Kriyoprezerve her satırı için, başlamadan önce aşağıdaki hazırlamak. <ol><li> 1 kiti CPA 240 l 4-iyi bir yemek, iyi </li><li> Aşağıdaki gibi 15 sperm dondurulması payet <ol><li> Şırıngaya yakın pamuk fişi ile 1 ml şırınga 0,25 ml payet bağlayın. Her saman, sonra IVF orta 8 cm (yaklaşık 160 ul), 1 cm hava yükleyin. Payet korunacak hattın adı ile etiketleyin. </li></ol></li></ol></li><li> LN 2 derin köpük kutusunda 15 cm olmalıdır. Kapağını kapatın. </li><li> Kriyoprezerve her satırı 2 erkek fareler Euthanize. Fareler, 10 ve 60 haftalık arasında olmalıdır. Her fare vas deferens ile birlikte iki kaudal epididymides çıkarın. </li><li> Önceden hazırlanmış 4-iyi bir çanak (adım 1.1) EBM içeren epididymides yerleştirin. </li><li> Her epididim 5-7 uzunlamasına keser ve nazik olunSperm dışarı itmek için her vas deferens sıkın. </li><li> 3-5 dakika oda sıcaklığında CPA ve epididim karışımı elle hafifçe sallayın. Bu sperm yüzmek için izin verecektir. </li><li> Önceden hazırlanmış saman kullanarak, sperm süspansiyonu 5 mm (yaklaşık 10 ul) aspirat ve ardından havanın 1 cm. </li><li> Toplam 15 payet kadar bu işlemi tekrarlayın. Yük ve en iyi sperm hayatta kalmak için 10 dakika içinde payet mühür. </li><li> Saman her iki tarafında dikkatli mühür ısıtın. Ilk sperm sütun donma spreyi payet yerleştirin. </li><li> 1 ml pipet kolu uzatmak için spreyi kitinin bir parçası olarak sağlanan takın. LN 2 teneke kutu kapağı delikten köpük kutusuna yerleştirin. 10 dakika için LN 2 dikey float spreyi izin verin. </li><li> LN2 spreyi bırakın. Sperm Kriyoprezerve ve uzun vadeli LN 2 depolama taşınmış olabilir. </li><li> Spreyi kullanılmamalıdırbir sonraki satıra kadar oda sıcaklığına kadar ısıtılır. </li></ol> 2. Fare süperovulasyon <ol><li> Genel olarak, genç dişi farelerde sperm vericisi olarak aynı arka plan kullanın. </li><li> Fare ~ 12 g (yaş 3-4 hafta) olmalıdır. </li><li> Hamile kısrak serum gonadotropini (PMSG) 5-10 IU ile bir intraperitoneal gerçekleştirin. </li><li> 46-48 saat bekleyin. </li><li> 5-10 IU insan koryonik gonadotropin (hCG) ile bir intraperitoneal gerçekleştirin. </li><li> HCG enjeksiyonu sonrasında aşağıda açıklandığı gibi Hasat oosit 14-16 saat. </li></ol> 3. Fare sperm ve IVF <ol><li> IVF önce çözülmüş olması için her saman için, en az bir saat için 37 ° C CO 2 inkübatör aşağıdaki hazırlamak ve dengelenmesi. <ol><li> Sperm inkübasyon çanak: bir 35 mm Petri kabında 90 ul bir damla yağ ile kaplı sperm tedavi orta (STM). </li><li> Kesme çanak: Yumurta toplama için en fazla 10 snuperovulated kadın, 3 ml IVF orta ile 35 mm Petri kabında. </li><li> IVF: IVF için 5 süperovulasyon kadın, IVF orta 500 ul (.. 10 süperovulasyon kadın kullanıyorsanız, örneğin, 2 kuyu gerekecektir) ile 4 sıra çanak iyi bir her grup için </li><li> Kullanılan 4-iyi IVF çanak her iyi yıkamak için 3 ml potasyum simpleks optimize orta (KSOM) ile bulaşık bir 35 mm Petri kabı: </li><li> Kültür çanak: 50 IVF yemeğin her kuyudan petrol embriyo kültürü ile kaplı KSOM orta ul damla bir 35 mm Petri kabında. </li></ol></li><li> LN 2 ile saman ve 2-3 dakika 37 ° C su banyosunda yer çıkarın. </li><li> Su banyosu saman çıkarın ve fazla suyu kaldırmak için silin. İlk kesilen pamuk fiş en yakın ilk hava sütunu yakın olun. Hala bir hava sütunu sperm sütun önce görünür olacaktır IVF orta saman kesin. </li><li> Sonra saman s ucuecond hava sütun, sperm sütun kesme önlemek için dikkatli olmak. 45 açıyla Kes - Bu sperm aspire çok daha kolay hale getirecek. </li><li> 200 ul pipet ile saman distal ucundan sperm süspansiyonu aspire. </li><li> STM damla merkezi sperm süspansiyonu yerleştirin ve 45-60 dakika boyunca 37 ° C% 5 CO 2 inkübatör inkübe çanak. </li><li> Sperm inkübasyon sırasında, kesme çanak içine süperovulasyon kadın siyon hatası teşrih. Cumulus-oosit kompleksleri (COC) bırakmadan, her yumurtalık gözyaşı 30 g iğne veya forseps kullanın. IVF orta kirli medya, ekstra yıkama adımlar için yapar gibi yağ ve kan kaçının. </li><li> Bir kez tüm siyon hatası yıkılarak, 5 kadın her IVF kuyuları Kombine oral kontraseptif aktarın. Süperovulasyon tarafından üretilen kombine oral kontraseptif sayısı çok bağımlı süzün. </li><li> STM damla çevreden sperm 10-15 ul toplayın ve bir IVF döller de yaklaşık 14-16 saat hCG injection. Her bir kuyunun kullanılan gübreleme tekrarlayın. Final sperm konsantrasyonu yaklaşık 1 milyon / ml olacaktır. </li><li> 4-6 saat 37 ° C% 5 CO 2 inkübatör Coculture sperm ve oosit. </li><li> Embriyoların gelişimi için 37 ° C% 5 CO 2 inkübatör KSOM damla en az 2 kez ve kültür KSOM ile yıkayın. </li><li> IVF oranı gece kültürden sonra kullanılan toplam oositlerin iki hücre sahne olan embriyolar gibi hesaplanmıştır. </li></ol> 4. Temsilcisi Sonuçlar 5 Charles Nehri fare kendilenmiş suşlarının sperm donduruldu. Koruma oranları taze sperm değerlendirmesi değerleri ile dondurulmuş sperm değerlendirme değerleri bölünmesiyle hesaplanır. Sperm Koruma oranları sırasıyla% 60.2,% 81.3,% 78.6,% 66.5,% 61.0 &#39;C57BL/6NCrl, 129S2/SvPasCrl FVB / NCrl, DBA/2NCrl ve BALB / cAnNCrl. Hızlı progresif motilite için koruma oranları% 47.3,% 69.4,% 57.0,% 52.8,% 54.1 C57BL/6NCrl, 129S2/SvPas<html> Crl, FVB / NCrl, DBA/2NCrl ve sırasıyla BALB / cAnNCrl (Şekil 1). Dondurulmuş çözülmüş sperm ile tüp bebek oranı% 55.7,% 26,4,% 87.3,% 87.8 ve C57BL/6NCrl yılında sırasıyla 26.2%, 129S2/SvPasCrl, FVB / NCrl DBA/2NCrl ve BALB / cAnNCrl (Şekil 2) . 1.5 yıllık bir dönemde, 49 GM fare hatları sperm dondurulması tarafından arşivlenir. Bu satırlar, daha sonra başarılı IVF (C57BL / 6, BALB / c, DBA / 1 ve 129Sv), 4 kadın suşları (Şekil 3) (canlı doğan yavrular tarafından tanımlandığı gibi) ele geçirilmiştir. <img alt="Şekil 1." src="/files/ftp_upload/3713/3713fig1.jpg" /> Şekil 1: Fareler ve Sperm Hareketliliği Hızlı Aşamalı Motilite Koruma Oranları <img alt="Şekil 2." src="/files/ftp_upload/3713/3713fig2.jpg" /> Farelerde Dondurulmuş çözülmüş Sperm Şekil 2. In Vitro Fertilizasyon 3713fig3.jpg &quot;/&gt; Şekil 3. GM Farelerde Dondurulmuş çözülmüş Sperm ile in vitro fertilizasyon

<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>

Yazarlar Charles River, bu kit üreticisi çalışanları.

1990 yılından bu yana,% 18 raffinose ve% 3 yağsız süt kullanarak temel sperm dondurma protokol farelerin birçok suşları güvenilir kanıtlanmış ve çok sayıda fare hatları 1,2,3,4,5,6,7 Kriyoprezerve edilmiştir . Donma ve çözülme süreci sırasında sperm hücrelerinin zarar görmesi başlıca nedenleri, buz oluşumu 8,9 ve reaktif oksijen türlerinin 10 ile ilişkili olduğu var. Dondurucu bir ortamda böyle bir monotiyogliserol olarak amino asitler gibi serbest radikal temizleyiciler ve indirgeyici ajanlar, takviyesinin araştırılmalı ve C57BL / 6 11,6 kendilenmiş suşları dahil olmak üzere donmuş-çözülmüş sperm ile tüp bebek oranı önemli ölçüde artırmak için kanıtlanmış oldu. İşbu Protokol, piyasada bulunan antioksidanlar ve buz blokerleri ile temel EBM optimize ederek, sperm dondurma ve IVF işlemi değiştirerek fare sperm dondurulması için yeni bir I • Cryo kiti geliştirdi. Sperm Koruma oranları(&gt;% 60) ve hızlı progresif motilite (&gt;% 45) I • Cryo kiti ile dondurulmuş beş kendilenmiş fare suşlarının sperm görüldü. 5 fare kendilenmiş suşları dondurulmuş çözülmüş sperm ile 2-hücreli aşamasında embriyo gelişimi oranı temel EBM 11,6 ile kıyasla belirgin bir şekilde düzeldi. Kiti kullanılarak, kullanıcının her bir hat için 2 erkek dondurma sperm tarafından konsantre bir sperm süspansiyonu oluşturur. Sadece dondurulması 15 payet olarak süreci hızlı, kolay ve kısa ya da uzun süreli depolama için daha az depolama alanı kaplar. Çoğu kez, fare hatları sadece 1 veya 2 payet çözülme tarafından kurtarıldı.

<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

Binlerce yeni farelerin genetik yapısı değiştirilmiş (GM) suşları transgenesis ve boşaltma teknolojilerin gelişiyle bu yana oluşturulmuştur. Bu değerli hayvanların çoğu, canlı hayvan olarak sadece acil durumlarda herhangi bir yedekleme planı ile var. Embriyo Kriyoprezervasyon bu yedekleme sağlar, ancak pahalı olduğunu, uzun bir prosedür olması, ve genel olarak, başarı için hayvanların çok sayıda gerektirir. Fare sperm,% 18 raffinose ve% 3 yağsız süt oluşan bir temel dondurucu ajan (CPA) ile başarıyla Kriyoprezerve olabilir keşfinden beri, sperm dondurulması, bu değerli suşları, arşivleme, dağıtım ve kurtarma için kabul edilebilir ve maliyet-etkin bir yöntem haline gelmiştir . Burada fare sperm dondurulması için yeni geliştirilen I • Cryo kiti göstermektedir. Kendilenmiş farelerin beş yaygın kullanılan suşlar bu kit kullanarak sperm dondurulur ve daha sonra ele geçirilmiştir. Sperm Yüksek koruma oranları (&gt;5 kendilenmiş fare suşlarının bu kit ile dondurulmuş sperm için% 60) ve kontrol (temel EBM) ile karşılaştırıldığında, hızlı progresif motilite (&gt;% 45) görüldü. Sperm IVF sonra iki hücre aşamasında embriyo gelişimi incelendiğinde tüm 5 fare suşlarının sürekli geliştirildi. 1.5 yıllık bir dönemde, 49 GM fare hatları I • Cryo kiti ile sperm dondurulması tarafından arşivlenen ve daha sonra IVF tarafından kurtarıldı.

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

Burada fare sperm dondurulması için yeni geliştirilen I • Cryo kiti göstermektedir. Sperm ile dondurulmuş çözülmüş İki hücreli evre embriyo gelişimi bu kit kullanımı ile 5 fare suşlarının sürekli geliştirildi. 1.5 yıllık bir dönemde, 49 genetiği değiştirilmiş fare hatları I • Cryo kiti ile sperm dondurulması tarafından arşivlendiği ve daha sonra başarılı IVF tarafından kurtarıldı.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

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

Nanyang Technological University

Research

JoVE Journal

Biology

39.5K Görüntüleme.  Charles River. Burada fare sperm dondurulması için yeni geliştirilen I • Cryo kiti göstermektedir. Sperm ile dondurulmuş çözülmüş İki hücreli evre embriyo gelişimi bu kit kullanımı ile 5 fare suşlarının sürekli geliştirildi. 1.5 yıllık bir dönemde, 49 genetiği değiştirilmiş fare hatları I • Cryo kiti ile sperm dondurulması tarafından arşivlendiği ve daha sonra başarılı IVF tarafından kurtarıldı.

Video: 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.

Farelerin Hasat Sperm ve Suni Tohumlama

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 ...

Biyoloji

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.

Plazmid DNA Qiagen Miniprep Kit kullanarak Bakteri kolonileri Arındırıcı

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.

İmmünohistokimya: Parafin Bölüm Vektör Labs Vectastain ABC Seti

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

Bio-Rad Total RNA Kiti Kullanarak Neuroprecursor Hücreler RNA Ekstraksiyon

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.

Sigma&#39;nın Özü-N-Amp Doku PCR Kit kullanarak Fare Doku hızlı Genotiplendirme

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.

Hamster Oosit ve Fare Embriyolar Toplama ve Kriyoprezervasyon

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.

Zebra balığı sperm Kriyoprezervasyon için bir Yüksek Verim Yöntem ve In Vitro Gübreleme

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.

Etilen Glikol Tabanlı Vitrifikasyon Fare Embriyolar Kriyoprezervasyon

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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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.

Yazılımı kullanarak ve soğutma aygıtlarının balık Sperm değerlendirmesi

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