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

이 작품은 스페인 정부의 드 Educación Y Ciencia (BIO 2006-11792)와 Generalitat 드 Catalunya (2005 - SGR00437)에 의해 지원되었다. 저자는 미디어를 준비하는 그들의 기술 지원을 마크 Puigcerver 및 Jonatan 루카스에게 감사를 표합니다. Servei D&#39; Estabulari의 모든 직원은 동물의 주택에 대한 인정, 그리고이 비디오의 일부를 녹화에서 자신의 추가적인 기여 특히 후안 호세 마틴과 하비에르 베니토입니다. NCB가 Ciência E Tecnologia과 SG는 스페인 정부의 드 Educación Y Ciencia의 동료입니다 포르투갈어 Fundação 파라의 동료입니다.

동물 보호 절차 동물과 바르셀로나, 스페인 Universitat Autònoma 인간 연구에 대한 윤리위원회의 승인 지침과 규정에 따라 실시되었다. I.의 Oocyte 컬렉션 <ol><li> 두꺼운 질 분비물 (postovulatory 방전)의 존재에 대한 골든 시리아 변형의, 여성 햄스터 (8-12주의 옛 Mesocricetus auratus)을 확인하고 선택합니다. </li><li> 60시간 나중에 PMSG 40 IU의 intraperitoneal 주사 (임산부 마레 세럼 생식 샘 자극 호르몬) hCG (인간 Chorionic 생식 샘 자극 호르몬)의 40 IU 다음으로 superovulate 선택한 여성을 유발. </li><li> 16 시간 이산화탄소 챔버의 hCG의 관리 후 여성의 햄스터를 안락사. </li><li> 로 비디오에 나타난 여성의 oviducts 격리하고 KSOM - H 매체의 드롭에게 전송할 수 있습니다. </li><li> 37 hyaluronidase (156 U / ML)의 드롭 입체 현미경 수란관 ampulla을 이간하여 oocyte - 큐뮬러스 단지를 수집 ° C. Oocytes은 약 5 분 큐뮬러스 세포에서 해방된 것입니다. </li><li> hyaluronidase의 oocytes을 들고 KSOM - H 매체에 두 번 그들을 씻으십시오. </li></ol> 마우스 배아의 수집을 위해, 여성 생쥐 (뮤스 musculus; 6-8주의 오래된)은 나중에 hCG 48 시간의 5 IU 다음과 남성 생쥐와 성관계를 PMSG 5 IU의 intraperitoneal 주사로 superovulate을 유도하고 있습니다. 암컷은 24-26에서 자궁 전위에 의해 희생되며, 44-46 또는 54-56시간 pronuclear, 2 셀 또는 4 셀 무대 배아, 각각의 obtention위한 hCG의 관리 후. 햄스터 oocytes에 대해 설명한 pronuclear 배아의 수집이 정확하게 수행됩니다. 2 셀 및 4 셀 단계에서 배아는 KSOM - H 매체 [6]로 oviducts 플러싱에 의해 저장됩니다. 참고 :이 프로토콜에 따라 약 20-30 oocytes / 배아 각 여성에서 수집한 수 있습니다. II. 솔루션을 동결 융해의 준비 <ol><li> KSOM - H (propilenglycol 1.5 M) 5 ML에 propilenglycol의 0.57 ML을 diluting로, 솔루션을 준비합니다. </li><li> 0.2052 g의 자당 (자당의 0.3 M)와 튜브에 KSOM - H 2 ML를 추가하여 해동 솔루션을 준비합니다. </li><li> 0.0684 g의 자당 (propilenglycol와 0.1 M의 자당 1.5 M)과 튜브에 솔루션 I 2 ML를 추가하여 냉동 솔루션을 준비합니다. </li></ol> 참고 : 솔루션 I 및 솔루션을 동결 융해가 그들의 사용하기 전에 준비를해야합니다. 그러나, 동결 융해 솔루션의 준비에 사용되는 자당의 지정된 금액을 사전에 가중치를하고 몇 개월 동안 상온에서 3 ML - 튜브에 저장되어있을 수 있습니다. III. 냉동 프로토콜 <ol><li> 전송 oocytes / KSOM - H 매체에서 솔루션 I의 드롭하고 실온에서 7~15분에 대해 품어 배아. </li><li> 한편, 그것이 동영상 (각 솔루션 / 밀짚 1 방울)에 나와있는 솔루션을 동결 융해 90 μl 방울을 배치하여 빨대를 로드할 90mm 페트리 접시를 준비합니다. </li><li> 냉동 솔루션 방울에 oocytes / 태아의 원하는 번호를 전송합니다. 참고 : 우리는 보통 5-30 oocytes 또는 태아 / 드롭 사이에 전송할 수 있습니다. </li><li> 입 제어 흡인 시스템에 빨대를 첨부하며이 순서를 따라 그것을 로드할 수 : 솔루션 I, 공기, oocytes, 공기, 해동 드롭, 공기 동결 드롭합니다. </li><li> 짚으로이로드되면, 첫 번째 드롭은 빨대와 물개이를 상단 끝에있는 폴리머에 도달 (솔루션 I) 도입까지 aspirating 유지. 열 인감 플라스틱 플러그와 밀짚이나 인감의 다른 쪽 끝을. </li><li> 프로그램 냉동고에 스위치와 냉동 프로그램을 선택합니다. 이 프로토콜에 사용되는 냉동이 프로그램은 원래 Lassalle 호텔 외 발행 냉각 속도에 따라 달라집니다. [7] : <ul><li> -7로 실온에서 2 ° C / 분 ° C 0 ° C / 10 분 분 시딩의 온도 평균 및 유도 수 있도록 (아래 참조) </li><li> -7에서 0.3 ° C / ° -30 분 ° C로 C 0 ° C / 2 분 분 온도 평균 수 있도록 </li><li> 35 ° C / 분, -30 ° C에서 -130 ° C로 </li></ul></li><li> 해동 솔루션의 드롭으로 빨대 부분이 바깥쪽을 향하게되어 있는지 확인하고, 프로그래밍 냉동실의 소유자의 슬롯에 빨대를로드합니다. 냉동 프로그램을 시작합니다. </li><li> 샘플 온도 -7 ° C 및 냉각이 보류 중이라에 도달하면, 수동 시딩을 수행합니다 : 액체 질소에 빠지게 포셉는 즉시 해결책을 해동의 방울이 들어있는 빨대의 일부분을 노출 약간 밖으로 냉동실의 소유자를 당겨하고, 포셉와 짚으로이 부분을 터치. 홀더 각 소유자와 각 밀짚을 반복하고 끝날 때까지 실행 냉동 프로그램을 보자. </li><li> 프로그램이 완료되면, 액체 질소의 소유자를 뛰어들다. </li><li> 에서 빨대를 제거표시 gobelet에게 전송 보유자합니다. </li><li> 마지막으로, 액체 질소 탱크 내부 gobelet를 저장합니다. </li></ol> IV. 프로토콜을 해동 <ol><li> 액체 질소 탱크에서 빨대를 제거합니다. </li><li> 40 초전에 실내 온도에 빨대를 유지합니다. </li><li> 40초 동안 30 ° C에서 물 목욕에 빨대를 적시게. </li><li> 짚을에서 플러그를 제거하거나 열 봉인 팁 잘라. </li><li> 35 mm 배양 접시에 짚을 비우기 부드럽게 냉동가 포함된 솔루션을 융해 두 방울을 섞는다. </li><li> 실온에서 15 분 혼합물에서 oocytes / 배아를 남겨주세요. </li><li> 37 추가 15 분 동안 KSOM - H의 새로운 드롭 oocytes / 배아를 전송 ° C. </li><li> Oocytes / 배아는 이제 더 이상 조작을위한 준비가되어 있습니다. </li></ol> 참고 : 6 단계와 7 단계 동안은 해동 oocytes / 배아는 천천히 rehydration로 인해 팽창되는가 나타납니다. V. 대표 결과 cryopreservation 프로토콜의 사용은 여기 햄스터 oocytes 약 95 %와&gt; 90 %, 85% 각각 pronuclear, 2 셀, 4 셀 단계에서 마우스 B6CBAF1 배아 80 %의 생존율에 결과를 발표했다. 마우스 배아는 해동 후 KSOM에 체외에서 양식하는 경우, 적어도 70-80% 최적의 문화 환경에 blastocyst 단계에 도달해야합니다. 그것은 당신이 각 냉동 실험에서 그 oocytes / 배아의 제어 그룹 (빨대)를 포함하는 것이 좋습니다 oocytes / 태아의 나머지 부분이 추가 조작에 사용되기 전에이 그룹을 녹여. 컨트롤 그룹에 대한 생존 속도가 예상보다 낮은 경우, 같은 많은 다른 빨대를 녹여. 이 두 번째 그룹의 생존 속도도 낮은 경우이 동결 많은에서 남은 빨대를 폐기하십시오.

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	<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>

이 프로토콜의 햄스터 oocytes와 마우스 배아가 성공적으로 cryopreserved 수로. 일단 냉동, oocytes / 배아가 무한정 액체 질소 탱크에 저장하고 원하는 어떤 시간이나 장소에서 복구할 수 있습니다. 이것은 필요한 때마다 사용하는 샘플을 거의 준비가 갖는 장점을 제공합니다. 한편,이 프로토콜은 또한 라이브 동물 식민지의 유지 관리를 위해 경제적이고 안전한 대안으로 가치 마우스 종자 (예 : 유전자 변형 라인 등)에서 배아 cryobanks를 생성하는 데 사용할 수 있습니다. 이 프로토콜은 햄스터 oocytes 및 하이브리드 B6CBAF1 마우스 배아를 cryopreserve하도록 최적화됩니다하는 것이 중요합니다. 다른 유전 배경과 마우스 배아 중 후 해동의 생존 요금의 차이는 치료가 각 종 또는 oocytes / 배아의 변형에 cryopreservation 조건을 평가하고 oocyte의 발생 전에 적절한 프로토콜을 확립하기 위해 이동해야하므로, 8,9을 설명했습니다 또는 배아 cryobanks. 햄스터 oocytes는 정자의 침투 시험 또는 정자의 염색체 분석 10, 또는 intracytoplasmic 정자 주입 (ICSI)에 초보자를위한 자료를 연습으로 인간 보조 생식 클리닉에서 사용할 수 있습니다. 한편, pronuclear 또는 2 셀 단계에서 마우스 하이브리드 B6CBAF1 배아가 배아 문화 미디어 또는 incubators의 적절한 기능의 품질을 확인하는 인간 보조 생식 센터 또는 연구소에서 사용할 수 있습니다.

<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

언제 위팅엄 배아와 oocytes 처음으로 성공적으로 30여 년 전에 cryopreserved되었습니다<i&gt; 외.</i&gt;<sup&gt; 1</sup&gt; 그리고 Wilmut<sup&gt; 2</sup&gt; 별도 Parkening, 마우스 배아는 ° C, 그리고 몇 년 후 -196에서 냉동 및 보관 수 있다고 설명<i&gt; 외</i&gt;.<sup&gt; 3</sup&gt;는 cryopreserved oocytes의 체외의 수정 (IVF)에 인한 사는 자식의 탄생을 보도했다. 이후, cryopreservation 기술의 사용이 급속도로 인간과 동물의 지원 복제의 관행과 동물 유전 자원 보존에 필수적인 요소가 될 확산되었습니다. 느린 동결 vitrification : 현재 oocytes과 배아를 cryopreserve하는 데 사용되는 두 가지 방법이 있습니다. 접근 방식의 다양한는 동물과 수천명의 아이들이 모두 기술과 수백만을 향상시키기 위해 노력하는 데 사용되었습니다 것은 cryopreserved 배아에서 태어난되었습니다. 그러나, cryopreservation 관련된 중요한 단점은 아직 얼음 결정 형성이 솔루션 효과​​ 및 삼투 충격 게시 - 해동 oocytes과 배아 여러 cryoinjuries을 일으킬 것 때문에, 극복해야합니다. 프로그램 냉동고와 느린 냉동은 일반적으로 화학적 독성과 삼투 충격와 관련된 cryoprotectants의 낮은 농도를 사용하는 장점을 가지고 있지만, 낮은 농도에서 얼음 결정 형성을 피하기 위해 자신의 능력이 제한됩니다. 느린 냉동은 또한 수동 또는 자동 시딩를 사용하여 피할 수 있어야합니다 supercooling 효과를 유도<sup&gt; 4</sup&gt;. vitrification 과정에서, cryoprotectants 높은 농도는 아이스 결정의 형성을 억제하고 물을 확정되는 glasslike vitrified 국가의 형성으로 이어질하지만, 확장되지 않습니다. 그러나, 때문에 사용 농도에 cyroprotectants의 독성에, oocytes / 배아는 액체 질소에 직접 샘플을 잠수함이 잠수하기 전에, 시간의 아주 짧은 기간과 최소 볼륨 솔루션 cryoprotectant 솔루션에 노출 수 있습니다<sup&gt; 5</sup&gt;. 그것도 고가의 장비 (프로그램 냉동고)가 필요하지 않습니다있는 매우 빠른 방법입니다 때문에 지난 10 년, vitrification 더 인기를 끌고있다. 그러나, 느린 냉동은 oocyte / 배아 cryopreservation에 가장 널리 사용되는 방법으로하고 있습니다. 이 동영상 기사에서 우리는 하이 포스트 - 해동 서바이벌 속도로 햄스터 oocytes를 수집하고 천천히 동결하는 방법을 단계별로, 보여줍니다. 동일한 절차도 성공적으로 동결과 preimplantation 개발의 다른 단계에 마우스 배아를 해동 적용할 수 있습니다.

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

이 동영상 기사에서 우리는 하이 포스트 - 해동 서바이벌 속도로 햄스터 oocytes를 수집하고 cryopreserve하는 방법에 대한 단계별 데모를 제시한다. 동일한 절차도 성공적으로 동결과 preimplantation 개발의 다른 단계에 마우스 배아를 해동 적용할 수 있습니다.

수집 및 햄스터 Oocytes 및 마우스 태아의 Cryopreservation

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

Nanyang Technological University

Research

JoVE Journal

Biology

16.9K 조회수.  Universitat Autonoma de Barcelona. 이 동영상 기사에서 우리는 하이 포스트 - 해동 서바이벌 속도로 햄스터 oocytes를 수집하고 cryopreserve하는 방법에 대한 단계별 데모를 제시한다. 동일한 절차도 성공적으로 동결과 preimplantation 개발의 다른 단계에 마우스 배아를 해동 적용할 수 있습니다.

비디오: 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.

마우스 Oocytes의 검색

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

연구

생물학

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.

마우스 Preimplantation 배아에서 줄기 세포 라인의

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

발생학

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.

유전자 변형 마우스의 고효율 생산을 위한 동결 해동 배아 사용

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

유전학

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.

미성숙한 고양이 오키트의 최소 볼륨 바이트화

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.

마우스 Oocyte의 Microinjection, 성숙과 Ploidy 평가

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.

에틸렌 글리콜 기반 Vitrification에 의한 마우스 태아의 Cryopreservation

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.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

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.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

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.

siRNA를 매개 음소거로 마우스 난 모세포의 감수 스핀들 평가

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.

Zebrafish Spermatogonia 전체 Testes 바늘의 cryopreservation 몰입 울트라-빠른 냉각

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 ...