Bu çalışma CIRM hibe # TR-1250, RT1-01.108 ve CL1-00.502 tarafından finanse edildi.

Not: Tüm hayvan prosedürleri IACUC veya muadili tarafından onaylanması gerekir. Tüm cerrahi ekipman ameliyattan önce sterilize edilmelidir. Steril eldiven, örtü ve gazlı bez kullanılmalıdır. 1. Ameliyat öncesi hazırlık <ol><li> 6 haftalık Muş MUSCULUS CbySmn.CB17-Prkdc SCID / J erkek fareler veya fare diğer bağışıklık suşu edinin. </li><li> Tüm cerrahi aletler, eldiven, gazlı bez ve sterilize edin. </li><li> Accutase enjekte edilecek hPSCs ayrışır. </li><li> Hücreleri saymak ve matrigel DMEM/F-12 1:1 seyreltilerek 20-30 uL başına 1.000.000 hücreleri tekrar süspansiyon. Bitirene kadar buz üzerinde hücreleri tutun. </li><li> Genelde bu Tripan mavi enjekte edilir deney aracılığıyla, bir çalışma yapmak için yararlı olduğunu not edin. Bu, herhangi bir hücre boşa önce olası sorunları belirlemeye yardımcı olacaktır. </li><li> Vivaryum olarak, kurumunuzun kabul prosedürlerine göre farenizi uyutmak. Deneylerde, biz bir Anseth kullanılanizofluran ile ÇSED makinesi. Fareler başlangıçta 1L/min oksijen ve% 3-4 izofluran ile bir indüksiyon odasına konuldu. Bir kez anestezi, 1L/min oksijen ve% 2-3 izofluran ile bir burun konisi kullanılmıştır. </li><li> Clippers ile karın Tıraş ve karın ortasına başlayarak ve dışa doğru saat yönünde çalışma, fare karın ön duvarı temizleyin. İlk kullanım Povidon-İyot çözeltisi% 70 etanol ile yıkayın. Çubuklar her zaman değişen, 3 kez tekrarlayın. Bir doku kültürü veya kesme kaput içinde hayvan sıcak tutmak için bir ısıtma pedi için hayvan aktarın. </li><li> Sadece kalça seviyesinin altında steril cerrahi makas ile cilt ve periton 1 cm longitudinal kesi olun. </li><li> Forseps ile periton tutarken, başka bir steril forcept ile sağ kalça aşağı doğru ulaşacak ve ekli testis ile birlikte beyaz yağ dokusu dışarı doğru çekin. </li><li> Steril gazlı bez testisler yerleştirin. </li><li> Tüberkülin veya Hamilton (1 cc) şırınga wi doldurunhPSCs enjekte edilecek th. Sizin gibi WA09 (ayrıca H9 olarak bilinir) gibi pluripotent olduğunu bildiğiniz bir kontrol hPSC satırı eklemek iyi bir fikir olduğunu unutmayın. Bu şekilde size enjeksiyon ya da cerrahi kusurlu olup olmadığını belirlemek için kullanabileceğiniz bir pozitif kontrole sahip. </li><li> Yavaşça testis kapsül şişmeye başlarsa durdurmak, herhangi bir büyük kan damarları uzak testis kapsülünün merkezi haline hPSCs (20-30 uL) enjekte edilir. </li><li> Hücrelerinin reflü önlemek için yavaş iğne çıkarılır. </li><li> Forseps kullanarak, karında orijinal konumuna testis ve yağ dokusu transferi geri. </li><li> 2 veya 3 emilebilen sütürlerle periton kadar kapatın ve autoclips ile cilde yakın. </li><li> Kurtarır ve 1-2 gün boyunca günde iki kez ameliyat sonrası analjezik bazı formunuz (vivaryum kabul ne bakınız) verilen kadar fare sıcak tutulmalıdır. Ile anestezi ne de analjezik ne tümör gelişmesine engel olduğunu unutmayın. </li><li> Th Monitör6-12 hafta süreyle tümör büyümesi için e hayvan. Çok nadir olarak, tümör önceden ila 6 hafta sonra, enjeksiyon boyutu 5 mm kadar uzayabilir, böylece hayvanlar izlemek için önemlidir. Bu durumda, hayvanlar ötenazi ve tümörler işlenmiş ve her zamanki gibi analiz edilmelidir. </li><li> Tümör palpe edilebilir ve yaklaşık büyüklüğü 5mm ulaştığında, fare uyutmak ve kabul hayvan protokol prosedürlere göre bu kurban. </li><li> Buna göre tümör ve belge çıkarın - fotoğraf, tedbir boyutu, tartın. </li><li> Küçük parçalar halinde tümörün kesin ve% 4 paraformaldehit çözüm düzeltin. Örnekleri kadar% 4 paraformaldehit Mağaza kesit, boyama ve analiz için bir patolog gönderilir. </li></ol> 2. Temsilcisi Sonuçları: Bu protokol yapıldığı zaman olarak tanımlanan ve hücre çizgisi enjekte pluripotent, bir palpabl, görsel bariz, tümör en az 12 hafta içinde oluşturmalıdır. WA09 gibi kurulan hPSC hatları için, biz genellikle bakın6 hafta içinde tümörler. IPSC hatlar için 8-10 hafta içinde tümör görmek olağandışı değildir. Bu prosedür doğru gerçekleştirildi emin olmak için, bir pozitif kontrol olarak, pluripotent olduğu bilinen bir çizgi ile farelere enjekte etmek için çok önemlidir. Tümörler genellikle çok heterojen bir görünüm ve birçok ekli kistleri (Şekil 1). Bir patolog tarafından tümör örneklerinin analizi her üç germ (Şekil 2) farklılaşmış dokuların göstermelidir. <img alt="Şekil 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> Şekil 1. Testis kapsül Tipik hPSC elde teratom. Tarif protokolü takip, bir milyon WA09 hücreleri, bir bağışıklık sistemi zayıflamış fare testis kapsül içine enjekte edildi. Altı hafta sonra bir teratom gözlendi. A) Teratoma fare çekilir. B) teratom resmi kadar kapatın. Heterojenite ve kist yapıları unutmayın. <img alt="Şekil 2" src = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; Şekil 2. Hematoksilin ve teratom gelen Eosin kesitlerin her germ tabakasından dokuları gösterir. Fiksasyonu takiben, teratom Hematoksilen ve Eosin ile kesitli ve lekeli idi. Bir patolog tarafından Analizi 3 germ her birinden hücrelerinin varlığını ortaya çıkarmıştır.

<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>

Çıkar çatışması ilan etti.

Burada sunulan yöntemin testis kapsül hPSCs gelen teratomlar doğurmanın çok güvenilir, kolay yol sağlar. Bu tekniğin birçok kritik parametre vardır. Özellikle, bir kontrol olarak pluripotent olduğu bilinmektedir hPSC hücre çizgileri enjekte etmek için önemlidir. Diğer önemli parametreler enjeksiyon ve tümör gözlenmesi arasındaki zaman aralığı vardır. WA09 gibi hücre hatları için, teratom 6-8 hafta dikkat edilmelidir. Yeni iPSC hatları için biz genellikle 10 hafta gerekli olduğunu bulmak. Başka bir endişe enjekte hücrelerin sayısıdır. Biz bir milyon hücre enjekte ama tahlil kolaylıkla daha az hücre ile yapılabilir. Buna ek olarak, enjeksiyon, orta önemlidir. Biz PBS veya DMEM/F-12 aksine hücreler, matrigel ve DMEM/F-12 bir 1:1 karışımı enjekte edildiğinde biz en iyi sonuçları almak bulabilirsiniz.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Accutase </td><td> Invitrogen </td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 113300-032 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

Pluripotent kök hücreler (PSC&#39;ler) bunlar her üç germ hücreleri içinde farklılık olabilir benzersiz özelliği var. Bu, bunların pek çok farklı hastalıkların tedavisi için potansiyel olarak değerli bir araçtır. Uyarılmış pluripotent kök hücreler (iPSCs) ve insan embriyonik kök hücreleri ile devam eden araştırma çıkışıyla (hESC) belirli bir hücre hattı pluripotent olduğunu kanıtlamanın deneyleri için bir ihtiyaç vardır. Germline iletim fare embriyonik kök hücre (Mesc) hatları 1,2,3 pluripotence göstermek için altın standart olmuştur. Bu testte kullanarak, araştırmacılar Mesc hattı germ hücreleri dahil olmak üzere 4 embriyo tüm hücre tipleri yapabilirsiniz gösterebilirsiniz. İnsan EKH germline iletimi için test edilemez yana insan ESC hatları 5,6 üretimi ile, bu hücrelerin pluripotence kanıtlamak için uygun bir test belli değildi. Bir taşıyıcı olarak, teratomlar assay anda pluripot göstermek için kullanılırinsan pluripotent kök hücrelerinin ğ (hPSCs) 7,8,9. Bu deneyi son zamanlarda, büyüteç altına alındı ​​ve yeni teknolojilerin etkin araştırılmaktadır rağmen, teratom tahlil günümüzde altın standart 7&#39;dir. Bu deneyde, söz konusu hücre, bir bağışıklık sistemi zayıflamış fare içine enjekte edilir. Hücrelerin pluripotent olduğunu, bir teratom sonuçta gelişecektir ve tümörün bölümleri her 3 germ 10 dokuların gösterecektir. Teratomlar deneyde, hPSCs fare farklı bölgelerde içine enjekte edilebilir. Veya bacak ya subkutan veya intramüsküler 11 içine; en yaygın enjeksiyon siteleri testis kapsül, böbrek kapsül, karaciğer içerir. Burada tümör büyümesi için site olarak testis kapsül kullanarak hPSCs gelen teratomların nesil için sağlam bir protokol tanımlamaktadır.

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

Teratoma Generation in the Testis Capsule

İnsan pluripotent kök hücreler (hPSCs) farklı hastalıkların sayısız tedavi potansiyeline sahiptir. Bu hücrelerin yardımcı vücutta herhangi bir hücre tipinde farklılaşabilmektedir olduğu yatmaktadır. Burada hPSCs arasında pluripotence göstermek için kullanılan deney teratomlar, açıklanmaktadır.

Testis Kapsül içinde teratom Üretimi

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 Görüntüleme.  Scripps Research Institute. İnsan pluripotent kök hücreler (hPSCs) farklı hastalıkların sayısız tedavi potansiyeline sahiptir. Bu hücrelerin yardımcı vücutta herhangi bir hücre tipinde farklılaşabilmektedir olduğu yatmaktadır. Burada hPSCs arasında pluripotence göstermek için kullanılan deney teratomlar, açıklanmaktadır.

Video: 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.

Dinlemede Making Sense IMAP Test Batarya:

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 ...

Tıp

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.

Ortak Bozuklukları de Novo Mutasyonlar, otizm ve şizofreni gibi tanımlayın Bir Strateji

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.

Fare Aortocaval Fistül Teknik Yönleri

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.

Prostat Karsinogenezde ve İyi Huylu Prostat Büyümesi ve Değerlendirilmesi için böbrek Kapsül Xenografting ve Derialtı Pelet İmplantasyonu

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.

Üretim ve Farelerde arteriyel Lezyonlarının 3-Boyutlu kantitasyonu Optik Projeksiyon Tomografi ile

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.

Yeni Nesil dizinleri için Toplama ve Tükrük DNA çıkarımı

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.

içinde Recombinase aracılı Kaset Borsası tarafından Rekombinant insan pluripotent kök hücreleri hızlı ve verimli Nesil<em&gt; AAVS1</em&gt; Odağı

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.

&quot;RE: İnce ilaçlar kullanılması&quot; İlaç repurposing Hipotez Üretim Sistemi

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.

Farelerde kapalı femur kırığı nesil: kemik şifa çalışması için bir Model

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.

Microwell Kültürü kullanılarak Testis'e Özgü Mimariye Sahip Domuz Testis Organoidlerinin Üretimi

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