Name: collection and cryopreservation of hamster oocytes and mouse embryos
Uploaded: 2009-03-27T00:00:00.000+00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Bu çalışma, İspanyol Ministerio de educacion y Ciencia (BIO 2.006-11.792) ve Generalitat de Catalunya (2005-SGR00437) tarafından desteklenmiştir . Yazarlar medya hazırlanmasında Marc Puigcerver ve Jonatan Lucas kendi teknik yardım için teşekkür etmek istiyorum. Servei d&#39;Estabulari tüm personeli, hayvan barınağı için kabul ve özellikle bu video kayıt ek katkı Juan Jose Martin ve Javier Benito. NCB Ciencia e Tecnologia ve SG, Portekizce Fundação para İspanyolca Ministerio de educacion y Ciencia bir adam bir adam .

Hayvan bakım ve prosedürleri, Barselona, ​​İspanya Universitat Autonoma Hayvan ve İnsan Araştırma Etik Komitesi tarafından onaylanmış kurallar ve düzenlemelere göre yapılmıştır. I. Oosit toplama <ol><li> Altın Suriye suşu kalın bir vajinal akıntı (postovulatory akıntı) varlığı için; dişi hamster (8-12 hafta eski Mesocricetus auratus) kontrol edin ve seçin. </li><li> 60 saat sonra intraperitoneal (Gebe Kısrak Serum gonadotropin) hCG (insan koryonik gonadotropin) 40 IU 40 IU PMSG superovulate için seçilen kadın neden. </li><li> Karbon dioksit odası hCG uygulanmasından 16 saat sonra dişi hamster Euthanize. </li><li> Siyon hatası videoda gösterildiği gibi kadın izole etmek ve KSOM-H orta bir damla aktarabilirsiniz. </li><li> 37 hiyalüronidaz (156 U / ml), bir damla stereoskopik mikroskop altında yumurtalık ampulla yırtarak oosit-kümülüs kompleksleri toplayın ° C. Oosit kümülüs hücreleri yaklaşık 5 dakika içinde serbest olacak. </li><li> Hiyalüronidaz gelen oosit pick up ve KSOM-H orta ve iki kez yıkayın. </li></ol> Fare embriyo toplanması için, dişi farelerde (Mus musculus; 6-8 hafta eski) daha sonra 5 IU hCG 48 saat takip ve erkek fareler ile evlendirilen PMSG 5 IU intraperitoneal superovulate indüklenen. Dişiler 24-26 servikal dislokasyon ile sakrifiye pronükleer, 2-hücre veya 4-hücreli aşamada embriyoların sırasıyla obtention hCG uygulandıktan sonra 44-46 veya 54-56 saat. Pronükleer embriyoların toplanması tam bir hamster oosit açıklandığı gibi yapılır. 2-hücre ve 4-hücreli aşamada Embriyolar KSOM-H orta [6] siyon hatası kızarma toplanır. Not: Bu protokol sonrasında yaklaşık olarak 20 ila 30 oosit / embriyoların her kadın alınacak . II. Dondurma ve çözme çözümler hazırlanması <ol><li> Propilenglycol KSOM-H (propilenglycol 1.5 M) 5 ml, 0.57 ml seyreltilmesiyle çözüm hazırlayın. </li><li> 0,2052 g sakkaroz (sakkaroz 0.3 M) ile bir tüp KSOM-H 2 ml ekleyerek çözülme çözüm hazırlayın. </li><li> 0,0684 g sakaroz (1.5 propilenglycol M ve 0.1 M sakkaroz) ile bir tüp çözümü ben 2 ml ekleyerek donma çözüm hazırlayın. </li></ol> Not: Çözüm I ve çözümler dondurma ve çözme sadece kendi kullanmadan önce hazır olmalıdır. Ancak, donma ve çözülme çözümler hazırlanması için kullanılacak sakaroz belirtilen miktarlarda avans ağırlıklı ve birkaç ay için 3 ml tüpler oda sıcaklığında muhafaza edilebilir. III. Donma protokol <ol><li> Transfer oosit / KSOM-H orta çözümü ben bir damla ve 7-15 dakika oda sıcaklığında inkübe embriyolar. </li><li> Bu arada, 90 video (1 damla her çözüm / saman) görüldüğü gibi, dondurma ve çözme çözümler ul damla koyarak payet yüklemek için 90 mm petri hazırlar. </li><li> İstenilen sayıda oosit / embriyo dondurma çözüm damla aktarın. Not: Biz genellikle 5 ila 30 oosit veya embriyo / bırak arasında transfer. </li><li> Ağız kontrollü aspirasyon sistemi bir saman takın ve bu sırada aşağıdaki yükü: çözümü, hava, oosit, hava, çözülme damla, hava ile dondurma bırak. </li><li> Saman yüklendikten sonra, saman ve mühürler Bu amaçla en sonunda polimer ulaşan ilk damla (çözüm) tanıtıldı kadar aspire tutun. Termo-plastik bir fiş ile saman veya mühür mühür diğer ucunu. </li><li> Programlanabilir dondurucu açın ve donma programı seçin. Bu protokolde kullanılan dondurma programı, Lassalle ve arkadaşları tarafından yayınlanan soğutma hızları dayanır. [7]: <ul><li> -7 Oda sıcaklığında 2 ° C / dak ° C 0 ° C / dk 10 dk tohum sıcaklık dengeleme ve indüksiyon için izin (aşağıya bakın) </li><li> -7 &#39;Den 0,3 ° C / dak ° C ila -30 ° C 0 ° C / dk 2 dk sıcaklık dengelenmesi için izin </li><li> 35 ° C / dak -30 ° C -130 ° C </li></ul></li><li> Çözülme çözüm damla ile saman parçası dışa karşı karşıya olduğunu emin olun, programlanabilir dondurucu sahibinin yuvaları payet yükleyin. Donma programını başlatın. </li><li> Örnek sıcaklığı -7 ° C ve soğutma beklemeye ulaştığında, elle tohumlama gerçekleştirin: sıvı azot içine atılmak forseps, hemen çözüm çözülme damla içeren payet bölümünü ortaya çıkarmak için, hafifçe dışarı dondurucu sahiplerine doğru çekin ve forseps ile payet bu dokunun. Sahibinin her tutucu ve her saman için tekrarlayın ve sonra donma program sonuna kadar işletilen sağlar. </li><li> Program tamamlandığında, sıvı nitrojen içinde sahiplerine dalıyoruz. </li><li> Payet Kaldıretiketli gobelet sahipleri ve aktarabilirsiniz. </li><li> Son olarak, bir sıvı nitrojen tankı içinde gobelet saklayın. </li></ol> IV. Protokol Çözülme <ol><li> Sıvı nitrojen tankı saman çıkarın. </li><li> 40 saniye boyunca oda sıcaklığında saman koruyun. </li><li> 40 saniye boyunca 30 ° C&#39;de su banyosunda saman bekletin. </li><li> Saman fişi çıkarın veya termo-mühürlü ucu kesmek. </li><li> 35 mm petri saman boşaltın ve hafifçe donma ve çözülme çözümler içeren iki damla karıştırın. </li><li> Oda sıcaklığında 15 dakika boyunca karışımı oosit / embriyoların bırakın. </li><li> 37 KSOM-H taze bir damla ilave bir 15 dakika için oosit / embriyo transferine ° C </li><li> Oosit / embriyoların şimdi manipülasyon için hazır. </li></ol> Not: 6. ve 7. adımları sırasında çözülmüş oosit / embriyoların yavaş rehidrasyon için nedeniyle şişer göreceksiniz. V. Temsilcisi Sonuçlar Kriyoprezervasyon protokol burada bir hamster oosit için yaklaşık% 95 ve&gt;% 90,% 85 ve fare B6CBAF1 embriyolar pronükleer, 2-hücre ve 4-hücreli aşamaları sırasıyla% 80, sağ kalım oranı sonuçlar bildirdi. Fare embriyoları çözme sonrası KSOM in vitro kültür, en az 70 ila% 80, en iyi kültür koşullarında blastosist aşamasına ulaşması gerekir. (Saman) ve oosit / embriyo dondurma her deneyde bir kontrol grubu yer almasını tavsiye ederiz oosit / embriyo geri kalanında daha fazla manipülasyonları için kullanılan önce bu grup Çözülme. Kontrol grubunda sağkalım oranları beklenenden daha düşük ise, aynı partinin diğerine saman Çözülme. Bu ikinci grup için sağkalım oranları da düşük ise, bu dondurma çok kalan payet atın.

<ol>
	<li>Whittingham, D. G., Leibo, S. P., Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+mouse+embryos+frozen+to+-196+and+-269+&#176;C.">Survival of mouse embryos frozen to -196 and -269 &#176;C.</a> Science. 178, 411-414 (1972).</li><li>Wilmut, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cooling+rate,+warming+rate,+cryoprotective+agent+and+stage+of+development+on+survival+of+mouse+embryos+during+freezing+and+thawing.">The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing.</a> Life Sci. 11, 1071-1079 (1972).</li><li>Parkening, T. A., Tsunoda, Y., Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+various+low+temperatures,+cryoprotective+agents+and+cooling+rates+on+the+survival,+fertilizability+and+development+of+frozen-thawed+mouse+eggs.">Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs.</a> J. Exp. Zool. 197, 369-374 (1976).</li><li>Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryobiological+principles+of+embryo+freezing.">Cryobiological principles of embryo freezing.</a> J. In Vitro Fert. Embryo Transf. 3, 3-9 (1986).</li><li>Vajta, G., Kuwayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cryopreservation+systems.">Improving cryopreservation systems.</a> Theriogenology. 65, 236-244 (2006).</li><li>Duselis, A. R., Vrana, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrieval+of+mouse+oocytes.">Retrieval of mouse oocytes.</a> JoVE. 3, (2007).</li><li>Lassalle, B., Testart, J., Renard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryo+features+that+influence+the+success+of+cryopreservation+with+the+use+of+1,2+propanediol.">Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol.</a> Fertil. Steril. 44, 645-651 (1985).</li><li>Ib&#225;&#241;ez, E., Grossmann, M., Vidal, F., Egozcue, J., Santal&#243;, J. <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+caprine+beta-lactoglobulin+transgenic+mouse+embryos.">Cryopreservation of caprine beta-lactoglobulin transgenic mouse embryos.</a> Cryobiology. 35, 290-298 (1997).</li><li>Dinnyes, A., Wallace, G. A., Rall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genotype+on+the+efficiency+of+mouse+embryo+cryopreservation+by+vitrification+or+slow+freezing.">Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing.</a> Mol. Reprod. Dev. 40, 429-435 (1995).</li><li>Tateno, H., Kamiguchi, Y., Mikamo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+freezing+and+thawing+method+of+hamster+oocytes+designed+for+both+the+penetration+test+and+chromosome+assay+of+human+spermatozoa.">A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.</a> Mol. Reprod. Dev. 33, 202-209 (1992).</li><li>Biggers, J., McGuinnis, L. K., Raffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+preimplantation+development+of+the+mouse+in+the+protein-free+KSOM.">Amino acids and preimplantation development of the mouse in the protein-free KSOM.</a> Biol. Reprod. 63, 281-293 (2000).</li></ol>

Bu protokol, bir hamster oosit ve fare embriyoları ile başarıyla Kriyoprezerve olabilir. Bir kez dondurulmuş, oosit / embriyoların sıvı nitrojen tanklarında süresiz saklanır ve istenilen herhangi bir yerde veya zamanda telafi edilebilir. Bu ihtiyaç duyulduğunda kullanmak örneklerin neredeyse hazır sahip olmanın avantajını sunar. Öte yandan, bu protokol de, canlı hayvan kolonileri bakımı, ekonomik ve güvenli bir alternatif olarak değerli fare suşları (transgenik hatları gibi), embriyo cryobanks oluşturmak için kul...

<table><tbody><tr><td>Material Name</td><td>Type</td><td>Company</td><td>Catalogue Number</td><td>Comment</td></tr><tr><td>French type mini-straw 0.25 ml</td><td></td><td>Mini-tub</td><td>13407/0010</td><td></td></tr><tr><td>hCG</td><td></td><td>Farma-Lepori, Spain</td><td>749036.4</td><td></td></tr><tr><td>Hyaluronidase</td><td></td><td>Sigma</td><td>H-3884</td><td></td></tr><tr><td>KSOM-H medium</td><td></td><td></td><td></td><td>Ref. [11]</td></tr><tr><td>Liquid Nitrogen </td><td></td><td>Air Liquide</td><td></td><td></td></tr><tr><td>Plastic plugs</td><td></td><td>Mini-tub</td><td>19046</td><td></td></tr><tr><td>PMSG</td><td></td><td>Intervet, Spain</td><td>1.614/8447</td><td></td></tr><tr><td>Propilenglycol</td><td></td><td>Fluka</td><td>82280</td><td></td></tr><tr><td>Sucrose</td><td></td><td>Merck</td><td>107687</td><td></td></tr><tr><td>Surgical Scissors</td><td></td><td>Aesculap</td><td>BC-060R; BG-061R</td><td></td></tr><tr><td>Thermo-sealer</td><td></td><td>SIZ</td><td>220</td><td></td></tr><tr><td>35 and 90 mm culture dishes</td><td></td><td>Nunc</td><td>153066; 150350</td><td></td></tr><tr><td>10 ml tubes</td><td></td><td>Greiner Bio-one</td><td>163160</td><td></td></tr><tr><td>Nitrogen tank</td><td></td><td>MVE, Cryogenics</td><td>XC 43/28</td><td></td></tr><tr><td>Programmable Freezer</td><td></td><td>Planner Products Ltd.</td><td>Kryo-10 Series III</td><td></td></tr><tr><td>Forceps</td><td></td><td>Aesculap</td><td>BD-331R; BD-053R; OC-020R</td><td></td></tr></tbody></table>

collection and cryopreservation of hamster oocytes and mouse embryos

Zaman Whittingham Embriyolar ve oositlerin ilk başarıyla, 30 yıldan daha uzun önce Kriyoprezerve edildi<i&gt; Ve ark.</i&gt;<sup&gt; 1</sup&gt; Ve Wilmut<sup&gt; 2</sup&gt; Ayrı Parkening fare embriyoları ° C ve birkaç yıl sonra -196 dondurulur ve saklanır olabilir tanımlanmıştır<i&gt; Ve ark.</i&gt;.<sup&gt; 3</sup&gt; Kriyoprezerve oositlerin in vitro fertilizasyon (IVF) kaynaklanan canlı yavrular doğum bildirdi. O zamandan beri, kriyoprezervasyon teknikleri kullanımı, insan ve hayvan üremeye yardımcı uygulama ve hayvan genetik kaynaklarının korunması konusunda önemli bir bileşeni haline hızla yayıldı. Yavaş dondurma ve vitrifikasyon: Şu anda, oosit ve embriyoların cryopreserve için kullanılan iki ana yöntem vardır. Kriyoprezerve embriyolar, hayvan ve binlerce çocuk her iki tekniğin ve milyonlarca artırmak için çok çeşitli yaklaşımlar denemek için kullanılmıştır doğmuştur. Ancak, dondurulması ile ilişkili önemli eksikliklerin hala buz kristali oluşumu, çözüm etkileri ve ozmotik şok sonrası çözülmüş oosit ve embriyolarda çeşitli cryoinjuries neden olacak gibi görünüyor bu yana, aşılabilir. Programlanabilir dondurucular Yavaş dondurma genellikle kimyasal toksisite ve ozmotik şok ile ilişkilidir cryoprotectants, düşük konsantrasyonlarda kullanılarak avantajı vardır, ama düşük konsantrasyonlarda buz kristali oluşumunu önlemek için yeteneği sınırlıdır. Yavaş dondurma, ayrıca manuel veya otomatik tohumlama kullanarak kaçınılmalıdır soğuma etkileri indükler<sup&gt; 4</sup&gt;. Vitrifikasyon süreçte, yüksek konsantrasyonlarda cryoprotectants buz kristallerinin oluşumunu engeller ve su katılaşmış olduğu bir glasslike vitrifiye devlet oluşumuna yol, ancak genişletilmiş değil. Ancak, kullanılan konsantrasyonlarda cyroprotectants toksisite nedeniyle oosit / embriyoları sıvı nitrojen içinde doğrudan örnekleri daldırarak önce, zaman çok kısa sürede ve en az bir ses çözümü dölveriminin çözüm maruz bırakılmamalıdır.<sup&gt; 5</sup&gt;. Pahalı ekipman (programlanabilir dondurucu) gerekli olduğu çok hızlı bir yöntem olması nedeniyle son on yıl içinde, vitrifikasyon daha popüler hale gelmiştir. Ancak, yavaş dondurma oosit / embriyo dondurulması için en yaygın kullanılan yöntem olmaya devam etmektedir. Bu video-makale-çözülme sonrası sağkalım oranları yüksek bir hamster oosit toplamak ve yavaş yavaş dondurma nasıl adım adım göstermektedir. Aynı işlem, başarılı bir şekilde dondurma ve preimplantasyon gelişimi farklı aşamalarında fare embriyoları Çözülme uygulanabilir.

Watch this Scientific Journal Video about Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos at JoVE.com

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Bu video-makale-çözülme sonrası sağkalım oranları yüksek bir hamster oosit toplamak ve cryopreserve nasıl bir adım-adım gösteri sunuyoruz. Aynı işlem, başarılı bir şekilde dondurma ve preimplantasyon gelişimi farklı aşamalarında fare embriyoları Çözülme uygulanabilir.

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 ...

collection-and-cryopreservation-of-hamster-oocytes-and-mouse-embryos

Research

JoVE Journal

Biology

16.9K Görüntüleme.  Universitat Autonoma de Barcelona. Bu video-makale-çözülme sonrası sağkalım oranları yüksek bir hamster oosit toplamak ve cryopreserve nasıl bir adım-adım gösteri sunuyoruz. Aynı işlem, başarılı bir şekilde dondurma ve preimplantasyon gelişimi farklı aşamalarında fare embriyoları Çözülme uygulanabilir.

Video: Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Fare Oosit alımı

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Biyoloji

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Fare Preimplantasyon embriyo kök hücre satırlarından türetme

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Gelişim Biyolojisi

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

Genetiği Değiştirilmiş Farelerin Yüksek Verimli Üretimi için Dondurulmuş Çözülmüş Embriyoların Kullanımı

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Genetik

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

Olgunlaşmamış Kedi Yumurtalarının Minimum HacimVitrifikasyonu

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Fare Oosit Mikroenjeksiyon, olgunlaşması ve ploidi Değerlendirmesi

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads 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 ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;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 valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

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 Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Serumsuz Tüm Embriyo Kültür Sistemde Fare Embriyonik Development

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

SiRNA aracılı susturma Mouse Oositlerinin içinde mayoz Mili Değerlendirmesi

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Zebra balığı Spermatogonia bütün testis iğne tarafından dondurma hızlı soğutma dalmış

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...