Name: germ cell transplantation and testis tissue xenografting in mice
Uploaded: 2012-02-06T12:00:00
Description: Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

著者の研究室からの作業はUSDA / CSREES / NRICGP（2007-35203-18213）によってサポートされている。NIH / NCRR（2 R01 RR17359-06）、NIH / NIEHS（1 R21 ES014856-01A2）とアルバータ州革新 - 健康ソリューション。

マウスではPART Aの生殖細胞移植 1。レシピエントマウスの準備<ol><li>受信者はドナー精巣細胞に（いずれかの遺伝的にドナーにマッチした、または免疫不全）、免疫学的に寛容でなければなりません。 </li><li>受信者のいずれか自然に精子を欠いている（例えば、W / W Vマウス）または内因性の生殖細胞が枯渇しなければなりません。生殖細胞の枯渇は、照射?...

<ol>
	<li>Brinster, R. L., Avarbock, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+of+donor+haplotype+following+spermatogonial+transplantation.">Germline transmission of donor haplotype following spermatogonial transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (24), 11303-11303 (1994).</li><li>Brinster, R. L., Zimmermann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+following+male+germ-cell+transplantation.">Spermatogenesis following male germ-cell transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (24), 11298-11298 (1994).</li><li>Nagano, M., Avarbock, M. R., Brinster, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+essential+for+self-renewal+and+expansion+of+mouse+spermatogonial+stem+cells.">Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (47), 16489-16489 (2004).</li><li>Oatley, J. M., Avarbock, M. R., Telaranta, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+genes+important+for+spermatogonial+stem+cell+self-renewal+and+survival.">Identifying genes important for spermatogonial stem cell self-renewal and survival.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (25), 9524-9524 (2006).</li><li>Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+transplantation+and+testis+tissue+xenografting+in+domestic+animals.">Germ cell transplantation and testis tissue xenografting in domestic animals.</a> Animal Reproduction Science. 89 (1-4), 137-137 (2005).</li><li>Dobrinski, I., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+germ+cells+from+rabbits+and+dogs+into+mouse+testes.">Transplantation of germ cells from rabbits and dogs into mouse testes.</a> Biology of Reproduction. 61 (5), 1331-1331 (1999).</li><li>Honaramooz, A., Snedaker, A., Boiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+from+neonatal+mammalian+testes+grafted+in+mice.">Sperm from neonatal mammalian testes grafted in mice.</a> Nature. 418 (6899), 778-778 (2002).</li><li>Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+testis+tissue+xenografting.">Recent developments in testis tissue xenografting.</a> Reproduction. 138 (2), 187-187 (2009).</li><li>Dobrinski, I., Rathi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+grafting+of+mammalian+testis+tissue+into+mouse+hosts.">Ectopic grafting of mammalian testis tissue into mouse hosts.</a> Methods in Molecular Biology. 139, 450-450 (2008).</li><li>Rodriguez-Sosa, J. R., Schlatt, S., Dobrinski, I., Seli, E., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+tissue+transplantation+for+fertility+preservation.">Testicular tissue transplantation for fertility preservation.</a> Fertility Preservation: Emerging Technologies and Clinical Applications. , 331-331 (2011).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+fate+and+seminiferous+tubule+development+in+bovine+testis+xenografts.">Germ cell fate and seminiferous tubule development in bovine testis xenografts.</a> Reproduction. 130 (6), 923-923 (2005).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+development+in+equine+testis+tissue+xenografted+into+mice.">Germ cell development in equine testis tissue xenografted into mice.</a> Reproduction. 131 (6), 1091-1091 (2006).</li><li>Kim, Y., Selvaraj, V., Pukazhenthi, B. <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+donor+age+on+success+of+spermatogenesis+in+feline+testis+xenografts.">Effect of donor age on success of spermatogenesis in feline testis xenografts.</a> Reproduction, Fertility, and Development. 19 (7), 869-869 (2007).</li><li>Arregui, L., Rathi, R., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenografting+of+adult+mammalian+testis+tissue.">Xenografting of adult mammalian testis tissue.</a> Animal Reproduction Science. 106 (1-2), 65-65 (2008).</li><li>Schlatt, S., Honaramooz, A., Ehmcke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+survival+of+adult+human+testicular+tissue+as+ectopic+xenograft.">Limited survival of adult human testicular tissue as ectopic xenograft.</a> Human Reproduction. 21 (2), 384-384 (2006).</li><li>Shinohara, T., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta1-+and+alpha6-integrin+are+surface+markers+on+mouse+spermatogonial+stem+cells.">beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5504-5504 (1999).</li><li>Kanatsu-Shinohara, M., Toyokuni, S., Shinohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD9+is+a+surface+marker+on+mouse+and+rat+male+germline+stem+cells.">CD9 is a surface marker on mouse and rat male germline stem cells.</a> Biology of Reproduction. 70 (1), 70-70 (2004).</li><li>Ryu, B. Y., Orwig, K. E., Oatley, J. 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D. <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+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. 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1。生殖細胞移植生殖細胞移植は、細胞集団中の精子幹細胞の存在（SSCS）の明確な確認のための唯一の機能的アッセイを提供しています。唯一のSSCはに家庭や基底膜でのSSCのニッチを植民地化とドナー由来の精子形成を開始することができます。生殖細胞移植は、前例のない形でSSCを勉強して操作することが可能になりました。技術は、SSCの4の遺伝子操作を介し?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>コラゲナーゼ（タイプIV） </td><td>シグマ</td><td> C5138 </td></tr><tr><td>トリプシン-EDTA </td><td>インビトロジェン</td><td> 25200-056 </td></tr><tr><td> DNaseIを</td><td>シグマ</td><td> DN25 </td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 31053-028 </td></tr><tr><td>トリパンブルー染色</td><td>インビトロジェン</td><td> 15250-061 </td></tr><tr><td>ナイロンメッシュのセルストレーナー</td><td> BDバイオサイエンス</td><td> 352340（40μmの） 352350（70μm） </td></tr><tr><td>ブスルファン</td><td>シグマ</td><td> B2635 </td></tr><tr><td>薄い壁ガラスキャピラリー</td><td>世界の精密機器</td><td> TW 100から3 </td></tr><tr><td> BD intramedic plyethyleneチューブ（PE100） </td><td> BD </td><td> CA-63018-725 </td></tr><tr><td>エチコン6から0シルク縫合糸</td><td>エチコン</td><td> 706G </td></tr><tr><td>創傷クリップ</td><td> BD </td><td> 427631 </td></tr><tr><td> Sigmacote </td><td>シグマ</td><td> SL2 </td></tr><tr><td> X-galを</td><td>シグマ</td><td> B4252 </td></tr><tr><td>フェロシアン化カリウム</td><td>シグマ</td><td> P9387 </td></tr><tr><td>フェリシアン化カリウム</td><td>シグマ</td><td> P3667 </td></tr><tr><td>塩化マグネシウム</td><td>シグマ</td><td> 208337 </td></tr><tr><td>デオキシコール酸ナトリウム</td><td>シグマ</td><td> D6750 </td></tr><tr><td> N、N-ジメチルホルムアミド</td><td>シグマ</td><td> D4551 </td></tr><tr><td> IGEPAL CA-630 </td><td>シグマ</td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

生殖細胞移植は、1994年1,2ペンシルベニア大学で博士ラルフBrinsterらによって開発されました。これらの画期的な研究では、レシピエント動物2でドナー由来の精子形成と精子の生産に不妊のレシピエントマウスの結果の精細管に肥沃なドナーマウスから生殖細胞のマイクロインジェクションを示した。レシピエント動物を1つ子孫にドナーハプロタイプのドナー由来の精子形成とトランスミッションの同定を可能に細菌性β-ガラクトシダーゼ遺伝子を有するドナーの男性の使用。驚くべきことに、精細管の内腔に移植後、移植した生殖細胞は精原細胞が3に位置している基底膜に内腔の区画から移動することができました。それは一般のみのSSCは、受信者の精巣におけるニッチと再確立する精子形成コロニーを形成することができることを受け入れられている。したがって、生殖細胞移植は、精巣の幹細胞ニッチを勉強すると推定される精原幹細胞を特徴づけるために機能的なアプローチを提供しています。現在までに、生殖細胞の移植は、基本的な幹細胞生物学を解明するために移植前4,5に生殖細胞の遺伝子操作を介してトランスジェニック動物を生成するために、セルトリ細胞の生殖細胞の相互作用6,7、SSCホーミングと植民地3を勉強するために使用されます8と同様に、SSCの自己複製と分化9,10。 生殖細胞の移植は大種11にも可能である。これらは、メインアプリケーションでは、妊孕性の保全、動物の個体数のエリート遺伝学の普及と、この手法で精子形成およびSSC生物学の研究で​​は、ロジスティックげっ歯類ではより困難かつ高価であるとしてトランスジェニック動物の世代です。 colonizatのマウスの結果の精細管に大きな種から生殖細胞の移植ドナー細胞と精子の拡大ではなく、系統的に遠い種12からの生殖細胞とレシピエントの体細胞コンパートメントの非互換性によるものであろう彼らの完全に分化におけるイオン。代替的アプローチは、その周囲の体細胞コンパートメントと一緒に大規模な種から生殖細胞の移植である。まず、免疫不全マウスの背部皮膚の下に移植した未熟な雄の精巣組織の小さな断片が受精有能な精子13の生産に完全に開発を生き残るためには、受けることができていることを、2002年に報告した。それ以来xenografting精巣組織は、多くの種で成功するために示されており、マウス14の大型動物の精巣発達と精子形成を研究する貴重な代替手段として浮上した。

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

xenografting生殖細胞移植と精巣組織のためのプロトコルが記述されています。生殖細胞移植レシピエント精巣におけるドナー由来の精子形成の結果、精原幹細胞（SSCS）を同定するための機能の再構成アッセイを表しています。 xenografting精巣組織は、レシピエントマウスの様々なドナー種の精巣発達と精子形成を再現します。

マウスでXenografting胚細胞移植と精巣組織

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

germ-cell-transplantation-and-testis-tissue-xenografting-in-mice

Research

JoVE Journal

Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

造血幹細胞の分離と移植（造血幹細胞）

生物学

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

アダルトゼブラフィッシュの全腎骨髄の移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

マウスにおける異所心臓移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

マウスにおける小腸移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

ヒトCD40活性化B細胞の生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

閉塞性気道疾患の発生研究のモデルとして使用されるマウスにおける異所性と同所性気管移植

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

プラナリアの固定化、部分照射し、組織移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

全細胞集団の単一細胞レベルでサイクリンBの分解を勉強

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...