この作品は、CIRM助成金＃TR-1250、RT1-01108、およびCL1-00502によって資金を供給された。

注：すべての動物の手順はIACUCまたは同等の承認を得なければならない。 すべての手術用機器は、手術前に滅菌しなければならない。滅菌手袋、ドレープとガーゼを使用する必要があります。 1。手術前の準備<ol><li> 6週齢ハツカネズミCbySmn.CB17-Prkdc SCID / J雄マウスまたはマウスの他の免疫不全の株を取得します。 </li><li>すべての手術器具、手袋、ガーゼを滅菌する。 </li><li> accutaseを注入するhPSCsを解離させる。 </li><li>細胞をカウントし、DMEM/F-12で1:1に希釈したマトリゲルで20から30μLあたり1,000,000細胞を再懸濁します。終了するまで氷上で細胞を維持する。 </li><li>しばしばそれはトリパンブルーが注入され、実験の経由実行を行うことが有用であることに注意してください。これは、任意のセルが浪費される前に潜在的な問題を特定するのに役立ちます。 </li><li>ビバリウムで、あなたの機関で受け入れられた手順に従って、マウスを麻酔。我々の実験で、我々はanesthを使用しイソフルランとESIAマシン。マウスは、最初に1L/min酸素と3から4パーセントイソフルランで誘導チャンバーに入れた。一度麻酔をかけ、1L/min酸素と2から3パーセントイソフルランとノーズコーンが使用されています。 </li><li>腹部の中心から開始して外側へ時計回りに働く、バリカンで腹部を剃り、マウスの腹部の前壁を清掃してください。最初の使用ポビドンヨード溶液を、次いで70％エタノールで洗浄する。綿棒を毎回変更し、3回繰り返します。組織培養や解剖フード内部の動物は暖かい保つために加熱パッドに動物を転送します。 </li><li>ちょうど股関節のレベル以下の滅菌外科用はさみで皮膚と腹膜を介して1センチメートル縦切開を行います。 </li><li>鉗子で腹膜を保持したまま、別の滅菌forceptで右股関節に向かって到達し、添付の精巣と一緒に白い脂肪組織を引き出します。 </li><li>滅菌ガーゼで精巣を配置します。 </li><li>ツベルクリンまたはハミルトン（1CC）をシリンジwiを埋めるhPSCsが注入される回。それはあなたがそのようなWA09（またH9）として知ら多能性であることがわかっているコントロールhPSC行を含めることをお勧めであることに注意してください。このようにあなたがあなたの注射や手術は、欠陥があったかどうかを判断するために使用できるポジティブコントロールを持っています。 </li><li>ゆっくり精巣カプセルが膨潤し始めた場合、停止、すべての主要な血管から離れた精巣カプセルの中心にhPSCs（20-30μL）を注入します。 </li><li>細胞の逆流を防ぐために徐々に針を外します。 </li><li>鉗子を用いて、腹部の元の位置に精巣と脂肪組織のバックを転送します。 </li><li> 2または3 reabsorbable縫合糸で腹膜を閉じて、オートクリップで皮膚を閉じます。 </li><li>マウスは1〜2日1日2回、それが回復するまで保温し、手術後に（あなたのビバリウムで受け入れられているものを参照してください）​​鎮痛剤のいくつかのフォームを与えられるべきである。 と麻酔も鎮痛剤のいずれも腫瘍の発達を妨げることに注意してください。 </li><li>モニター番目6-12週間の腫瘍成長のためのeの動物。非常にまれに、腫瘍は前に6週間、注射後のサイズは5mmに成長しないかもしれません、したがって、それは動物を監視することが重要です。この問題が発生した場合、動物を安楽死させ、腫瘍が処理され、いつものように分析する必要があります。 </li><li>腫瘍が触知され、サイズも約5ミリメートルに達すると、マウスを麻酔し、受け入れた動物プロトコルの手順に従って、それを生け贄に捧げる。 </li><li>それに応じて腫瘍やドキュメントを削除 - 写真、メジャーサイズ、重量を量る。 </li><li>小さな部分に腫瘍を切り取り、4％パラホルムアルデヒド溶液で固定します。サンプルまでの4％パラホルムアルデヒドでストアが切片、染色と分析のために病理学者に送信されます。 </li></ol> 2。代表的な結果： このプロトコルが実行されると前述のように、注入された細胞株は多能性であり、明白な、視覚的に明らかな腫瘍は最大で12週間以内に形成されなければならない。 WA09のような確立されhPSC行のために、私たちは一般的に参照してください。6週間以内に腫瘍。 IPSCの行のためには8-10週間以内に腫瘍を見ることも珍しくありません。これにより、プロシージャが正しく実行されていることを確認するために、ポジティブコントロールとして、多能性であることが知られている行をマウスに注入することが非常に重要です。腫瘍は通常、非常に不均一を見て、多くの添付嚢胞（図1）を持っています。病理医による腫瘍サンプルの分析はすべての3つの胚葉（図2）から分化した組織が表示されるはずです。 <img alt="図1" src="/files/ftp_upload/3177/3177fig1.jpg" /> 図1精巣カプセルの典型的なhPSC派生した奇形腫。 記述されたプロトコルに続いて、100万WA09細胞は、免疫妥協マウスの精巣カプセルに注入した。 6週間後に奇形が観察された。 A）奇形は、マウスから引き出された。 B）奇形腫の画像をクローズアップ。異質性と嚢胞構造を注意してください。 <img alt="図2" srC = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; 2。奇形腫から、ヘマトキシリンおよびエオシン染色した切片は、それぞれの胚葉由来の組織を示す図 。 固定後、奇形腫は、断面のヘマトキシリン​​およびエオシンで染色した。病理学者による分析は、3つの胚葉のそれぞれからの細胞の存在を明らかにした。

<ol>
	<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines.">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309, 255-255 (1984).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 78, 7634-7634 (1981).</li><li>Downing, G. J., Battey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+assessment+of+the+first+20+years+of+research+using+mouse+embryonic+stem+cell+lines.">Technical assessment of the first 20 years of research using mouse embryonic stem cell lines.</a> Stem Cells. 22, 1168-1168 (2004).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1145 (1998).</li><li>Reubinoff, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18, (2000).</li><li>Muller, F. J., Goldmann, J., Loser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6, 412-412 (2010).</li><li>Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+Setting+standards+for+human+embryonic+stem+cells.">Stem cells. Setting standards for human embryonic stem cells.</a> Science. 300, 913-913 (2003).</li><li>Lensch, M. W., Schlaeger, T. M., Zon, L. I., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+assays+with+human+embryonic+stem+cells:+a+rationale+for+one+type+of+human-animal+chimera.">Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera.</a> Cell Stem Cell. 1, 253-25 (2007).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+development+from+human+embryonic+stem+cells+after+injection+into+immunodeficient+mice.">Organized development from human embryonic stem cells after injection into immunodeficient mice.</a> Stem. Cells. Dev. 13, 421-421 (2004).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+embryonic+stem+cell-derived+teratomas+for+the+assessment+of+pluripotency.">Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.</a> Curr Protoc Stem Cell Biol. Chapter 1, Unit1B 4-Unit1B 4 (2007).</li></ol>

利害の衝突が宣言されません。

ここで紹介する方法は、精巣カプセルでhPSCsから奇形を発生させる信頼性の高い、簡単な手段を提供します。この手法では、いくつかの重要なパラメータがあります。特に、それはコントロールとして多能性であることが知られているhPSC細胞株を注入することが重要です。他の重要なパラメータは、注入し、腫瘍の観測の間の時間間隔が含まれています。 WA09のような細胞株では、奇形腫は6-8週間で観察されるべきである。新しいIPSCの行のために私たちはしばしば、10週間が必要とされることがわかります。もう一つの懸念は、注入された細胞の数です。我々は100万セルを挿入しますが、このアッセイは、簡単に少ない細胞で行うことができます。さらに、注入媒体は重要である。我々は、PBSまたはDMEM/F-12とは対照的に、細胞は、マトリゲルとDMEM/F-12の1:1混合物に注入されたとき我々は最良の結果を得ることがわかります。

<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> Accutase </td><td>インビトロジェン</td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td>インビトロジェン</td><td> 113300-032 </td></tr><tr><td>マトリゲル</td><td> BDバイオサイエンス</td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

多能性幹細胞（PSCの）彼らはすべての3つの胚葉由来の細胞に分化することができるユニークな特性を持っています。これは、それらのさまざまな疾患の治療のための潜在的に貴重なツールとなっています。ヒト胚性幹細胞（ヒトES細胞）の誘導多能性幹細胞（性IPSC）と継続的な研究の出現により、特定の細胞株は多能性であることを示すことができるアッセイの必要性がある。生殖系列伝達は、マウス胚性幹細胞（MESC）線1,2,3のpluripotenceを実証するためのゴールドスタンダードとされています。このアッセイを使用して、研究者は、MESCラインが生殖細胞4を含む胚内のすべての種類の細胞を作ることができることを示すことができる。ヒトESCが生殖系列伝達のためにテストすることはできませんので、人間のESC線5,6の生成では、これらの細胞のpluripotenceを証明するために適切なアッセイは不明であった。代理として、奇形腫アッセイは現在pluripotを実証するために使用されているヒト多能性幹細胞のency（hPSCs）7,8,9。このアッセイは最近、精査の下に来ており、新しい技術が積極的に検討されていますが、奇形腫アッセイは、現在のゴールドスタンダードは7です。このアッセイでは、問題の細胞は、免疫妥協マウスに注入される。細胞は多能性である場合、奇形腫は、最終的に開発し、腫瘍のセクションでは、全3胚葉10からの組織が ​​表示されます。奇形腫アッセイでは、hPSCsは、マウスのさまざまな領域に注入することができます。皮下又は筋肉内11のいずれか、または足に、最も一般的な注射部位は精巣カプセル、腎臓被膜、肝臓が含まれています。ここでは、腫瘍の成長のためのサイトとして精巣カプセルを使用してhPSCsから奇形腫を生成するための堅牢なプロトコルを記述します。

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

ヒト多能性幹細胞（hPSCs）は、様々な疾患の無数を治療する可能性があります。これらの細胞のユーティリティは、身体内の任意の細胞型に分化することができるという事実にある。ここではhPSCsのpluripotenceを実証するために使用される奇形腫アッセイを、説明します。

精巣カプセル内奇形腫の発生

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

teratoma-generation-in-the-testis-capsule

Research

JoVE Journal

Medicine

20.6K ビュー.  Scripps Research Institute. ヒト多能性幹細胞（hPSCs）は、様々な疾患の無数を治療する可能性があります。これらの細胞のユーティリティは、身体内の任意の細胞型に分化することができるという事実にある。ここではhPSCsのpluripotenceを実証するために使用される奇形腫アッセイを、説明します。

ビデオ: Teratoma Generation in the Testis Capsule

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAPのテストバッテリー：リスニングの意味を理解する

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

マウスAortocaval瘻の技術的な側面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

前立腺発がんおよび良性前立腺過形成の評価のための腎皮膜異種移植および皮下ペレット移植

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

次世代シーケンシングのための収集と唾液DNAの抽出

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

中リコンビナーゼ媒介カセット交換による組換えヒト多能性幹細胞の迅速かつ効率的な生成<em&gt; AAVS1</em&gt;軌跡

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

薬物転用仮説生成の使用」RE：罰金薬「システム

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

マウスで閉じた大腿骨骨折の生成: 骨の治癒を研究するためのモデル

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

マイクロウェル培養を用いた精巣特異的建築を用いたブタ精巣オルガノイドの生成

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...