EKİPMAN <ul><li> Doku kültürü inkübatör, 37 ° C,% 5 CO 2 </li><li> Santrifüj </li><li> Set micropipettors (100 100μL) </li><li> Su banyosu, 37 ° C </li><li> Su banyosu, 90 ° C buhar su kaynağı olarak </li><li> Faz kontrast mikroskobu </li></ul> İLK ADIM Colcemid ile hücrelerin tedavisi Bu prosedürde,% 20 nakavt serum replasmanı (KSR, Gibco) ve 8ng/mL, ile desteklenmiş, mitomisin C (Sigma) ile inaktive ve DMEM/F12 (Invitrogen) muhafaza fare embriyonik fibroblastlar (MEF) üzerine kültür H9 hESC hattı fibroblast büyüme faktörü (FGF-2, Ar-Ge). Metafaz yayılır, çok sayıda elde etmek için, birçok bölünen hücreleri vardır yüksek mitotik indeksi ile kültürlerin kullanılması tavsiye edilir. <ol><li> Inkübatör kültür şişesi alın ve laminer akış, son bir 0,1 mg / ml konsantrasyonda Colcemid eklemek. </li><li> Inkübatör balon dönün ve 3 saat bekleyin inhibitörü, kromozomlar yoğunlaşmış ve mikroskop altında kolayca görüntülenebilmekte metafaz, mitoz tutuklama neden olabilir. </li></ol> Hücrelerin Tespit <ol start="3"><li> Orta çıkarın ve hücrelerin yüzeyini kaplayan tripsin / EDTA% 0.05 &#39;yeterli eklemek. Hücreleri oda sıcaklığında 5 dakika inkübe edin ve sonra serum içeren kültür ortamı ekleyerek veya sadece hücreler serum ekleyerek ya enzim inaktive etmek için izin verin. NOT: iyi metafaz yayılır elde etmek için tek bir hücre süspansiyonu elde edilir emin yapmak için çok önemlidir. </li><li> 15 ml santrifüj tüpüne ve 5 dakika boyunca 122 g santrifüj hücrelere aktarın. </li><li> 200 ml hakkında bırakarak süpernatantı atın ve hafifçe videoda gösterildiği gibi tüp, dokunarak pelet tekrar süspansiyon haline getirin. Hücrelerin iyi ayrışmış, bu nedenle çözüm kümeleri görselleştirmek emin olun. </li><li> Sonra 5 ml hipotonik çözüm (KCl 75mm) 37 önceden ısıtılmış ° C, yavaş yavaş, aşağıdaki açıklamayı olduğu gibi tüp duvar eklemeniz gerekecektir İlk tüp invert hipotonik bir çözüm hemen sonra hücrelerin bir arada kalan 2 ml ekleyin ve 37 sıcak tutmak ° C 15 dakika boyunca yatay konumda, 3 ml ekleyin. NOT: Bu noktada hipotonik çözüm hücresel birimde bir artışa neden ve kromozomlar ayıklayabilir yardımcı olacaktır. Inkübasyon süresi, iyi bir kromozom elde etmek için önemlidir. Zamanlaması çok uzun ise, hücre zarı, çok erken ve kromozomlar kaybolur patlayabilir. Eğer çok kısa olursa, hücre zarı bozabilir çünkü kromozom yayılır elde etmek zor olabilir. </li><li> Fiksatif solüsyonu (metanol / asetik asit buzul 03:01) 3 damla ekleyin, 5 dakika ara vermeden 122 g hücreleri ve santrifüj fiksatif çözüm karıştırmak için tüp nazikçe çevirin. NOT: fiksatif solüsyon taze yapılmalıdır. NOT: malzeme kaybını önleyerek, hücrelerin yıpratma önlemek için ara vermeden santrifüj kullanmak önemlidir. </li><li> 200 ml hakkında bırakarak süpernatantı atın ve yavaşça tüpün alt dokunarak pelet tekrar süspansiyon haline getirin. NOT: Yine, hücrelerin iyi ayrışmış, bu nedenle çözüm kümeleri görselleştirmek emin olun. </li><li> Tüp döndürmek ederken, yavaş yavaş, fiksatif çözüm tüp duvar ilk 3 mL ekleyin. Hücrelerin karışımı, ve daha sonra tüp duvar hücreleri aşağı çekmek için 2 mL fiksatif çözüm daha eklemek için yatay pozisyonda Tüpü ters çevirin. 4 ° C gecede Stok. NOT: sabit hücreler, 4 ay için sabitleştirici çözüm saklanabilir ° C Kendi şişmiş durumda hücrelerini koruyarak yanı sıra, fiksatif çözüm lipitleri ve denatüre proteinlerin ortadan kaldırır. Bu olaylar sonucunda, kromozom kolayca yayılmasını, hücre zarının kırılgan yapmak ve. </li></ol> CAM KAYDIRAKLARI TEMİZLİK Kromozom yayılır slaytlarınız düzgün bir şekilde temizlenir veya bazı çekirdeklerin kaybedebilirsiniz ve aynı zamanda metafaz ki emin olun başlamadan önce. Eğer herhangi bir hibridizasyon tekniği 9 denemek istiyorsanız bu adımı yanı sıra önemlidir . <ol><li> Yeri oda sıcaklığında en az 3 saat 6M HCl içeren bir beher slaytlar. </li><li> Bu süreden sonra, 10 dakika boyunca çalışan musluk suyu slaytlar yıkayın. </li><li> Slaytları distile su ile çalkalayın ve oda sıcaklığında kurumaya bırakın. </li><li> Şimdi slaytlar% 96 etanol içinde saklanan ve kullanmak için bir havsız hemen önce kurutulmuş olabilir. </li></ol> NOT: situ hibridizasyon yöntemleri için, slaytlar, en fazla 2 gün boyunca% 96 etanol içine saklanabilir olamaz İkinci adım Çamaşır hücreler <ol start="10"><li> Ertesi gün, 5 dakika ara vermeden 122 g hücreler santrifüj. </li><li> D200 ml bırakarak süpernatant iscard. Nazikçe tüp dönerken tüp tüp duvar sonra yavaş yavaş taze fiksatif solüsyonu 5 ml (metanol / asetik asit buzul 03:01) eklemek Flicking pelletini tekrar. Bu yıkama üç yıkama olmak üzere toplam 2 kez daha adımı yineleyin. NOT: fiksatif solüsyon taze yapılmalıdır. </li></ol> Slaytlar hazırlanması <ol start="12"><li> (Bkz. Şekil 1) hücre pelletini homojenize ve slaytlar hücrelerin yeterli bir yoğunluk elde etmek için çözüm son santrifüj aşamada yeterli miktarda bırakın. </li><li> 20-30 mcL hücre süspansiyonu, temiz ve kuru bir slayt yüzeye çok yakın ve paralel hareket pipet üzerine ekleyin. Fiksatif çözüm buharlaşır gibi, slayt yüzeyi grenli olur. </li><li> Hemen havaya uçurmak hücrelerin neden 30 saniye süreyle sıcak su, buhar (90 °) içine, slayt maruz yüzü yukarı. NOT: Şu anda, asetik asit konsantrasyonu artmış ile sonuçlanan, metanol, fiksatif çözüm buharlaşır, su ile birlikte yayılan kromozom uyarır. </li><li> Açabildiği gibi, hücre dağılımı (bkz. Şekil 1) iyi olup olmadığını kontrol etmek için bir faz kontrast mikroskobu bakın. </li><li> Şimdi slaytlar G-bantlama (Giemsa), FISH, SKY veya CGH gibi yaklaşımlar sitogenetik kromozom seti kontrol etmek için kullanılmaya hazır. </li></ol> ÖNEMLİ İPUÇLARI Nasıl tercih analiz edilmesi için yeterli bir metafaz Yuvarlak ve izole metafaz (Şekil 2B) seçmek, iki veya daha fazla kromozom kazanç birlikte karışık veya başlattı kromozom tarafından oluşturulan kromozom kaybı yayılır yanlış anöploidiler dikkate kaçınmak olabilir. Yani, bazı kromozom (Şekil 2A ve 2B) tarafından gizli olabilir çünkü çekirdekleri yakın metafaz seçerek kaçının. Ayrıca, yuvarlak metafaz bakmak ve herhangi bir kromozom metafaz (Şekil 2C) ayrılmış olduğundan emin olun. Besleyici hücreleri ve hESC karyotip G-bantlama ve SKY analizi için, besleyici hücreler bölünerek devlet (mitomisin C veya ışınlama ile inaktif) altında değildir ve metafaz nüfusunun parçası olmayacaktır çünkü besleyici hücreler hESC kültür varlığı, sonuçları ile müdahale değildir. Ancak,, interfaz çekirdekleri FISH tekniğinin uygulanması için, besleyici hücrelerin koloniler FISH analizi için trypsinization önce elle hasat hESC nüfus ayırmak için tercih edilir. <img border="0" src="/files/ftp_upload/1512/1512fig1tmb.jpg" alt="Şekil 1" /> Şekil 1: slayt uygun hücre yoğunluğu nasıl biliyoruz. 10x objektif faz kontrast mikroskop görüntülerini. (A) hücre yoğunluğu çok yüksek. Ok çok yakın çekirdekleri yayılır metafaz işaret etmektedir. (B) iyi bir hücre yoğunluğu ile bir slayt. Çevreler, çekirdekleri veya diğer metafaz izole metafaz yayılır göstermektedir. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1512/1512fig1.jpg">buraya tıklayınız</a> . <img border="0" src="/files/ftp_upload/1512/1512fig2tmb.jpg" alt="Şekil 2" /> Şekil 2: Nasıl analiz edilmesi için yeterli bir metafaz seçin. Kromozomlar DAPI ve 100x objektif bir floresan mikroskobu ile elde edilen görüntüler ile boyandı. (A) (B) çekirdekleri yakın metafaz kaçının (oklarla gösterilen) (C) ve mevcut kromozom ayrılmış (daire) yuvarlak değil metafaz. (D) yuvarlak metafaz analiz edilmesi için iyi bir metafaz. Lütfen Şekil 2&#39;de büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1512/1512fig2.jpg">buraya tıklayın</a> .

<ol>
	<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 (5391), 1145-1145 (1998).</li><li>Draper, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+gain+of+chromosomes+17q+and+12+in+cultured+human+embryonic+stem+cells.">Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.</a> Nat Biotechnol. 22 (1), 53-53 (2004).</li><li>Maitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+alterations+in+cultured+human+embryonic+stem+cells.">Genomic alterations in cultured human embryonic stem cells.</a> Nat Genet. 37 (10), 1099-1099 (2005).</li><li>Mitalipova, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+the+genetic+integrity+of+human+embryonic+stem+cells.">Preserving the genetic integrity of human embryonic stem cells.</a> Nat Biotechnol. 23 (1), 19-19 (2005).</li><li>Buzzard, J. J., Gough, N. M., Crook, J. M., Colman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype+of+human+ES+cells+during+extended+culture.">Karyotype of human ES cells during extended culture.</a> Nat Biotechnol. 22 (4), 381-381 (2004).</li><li>Caisander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+integrity+maintained+in+five+human+embryonic+stem+cell+lines+after+prolonged+in+vitro+culture.">Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture.</a> Chromosome Res. 14 (2), 131-131 (2006).</li><li>Imreh, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+conditions+favoring+selection+of+chromosomal+abnormalities+in+human+ES+cells.">In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.</a> J Cell Biochem. 99 (2), 508-508 (2006).</li><li>Robin-Wesselschmidt, J. L., Loring, J. F., Robin-Wesselschmidt, J. L., Robin-Wesselschmidt, S. c. h. w. a. r. t. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Stem Cell Manual. , 59-59 (2007).</li><li>Rooney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cytogenetics. , (2001).</li><li>Heslop-Harrison, P., Schwarzacher, T., Heslop-Harrison, P., Schwarzacher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical in situ hybridization. , 51-51 (2000).</li></ol>

The authors have nothing to disclose.

Kromozom yayılır hazırlık, G-bantlama ve BALIK, SKY ve CGH gibi daha sofistike teknikler olarak rutin teknikleri ile embriyonik kök hücrelerin genetik bir durum başarılı bir analiz için önemli bir adım. Bu prosedür, hücre döngüsü uzunluğu bağlıdır colcemid kuluçka döneminde değişen birçok hücre tipleri için uygulanabilir. (EB) embriyoid organları için bu süre 6 saate kadar ise embriyonik kök hücre kolonileri için, biz 3 saat bekleyin. <p class="jove_cont...

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Techno Plastic Products</td><td>91015</td><td></td></tr><tr><td>Colcemid Karyo MAX</td><td>GIBCO, by Life Technologies</td><td>15212-012</td><td></td></tr><tr><td>EDTA</td><td>Isofar</td><td>721</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Isofar</td><td>100</td><td></td></tr><tr><td>Methanol</td><td>Isofar</td><td>208</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Merck &amp;amp; Co., Inc.</td><td>104.931.000</td><td></td></tr><tr><td>Slide</td><td>Bioslide</td><td>7105-1</td><td></td></tr><tr><td>Tripsin</td><td>Sigma-Aldrich</td><td>T4799</td><td></td></tr></tbody></table>

chromosomal spread preparation of human embryonic stem cells for karyotyping

Insan embriyonik kök hücreleri (hESC) istikrarlı bir diploid karyotip 1 sunmak için gösterilmiş olsa da, pek çok çalışmada, kültür koşullarına bağlı olarak bütün ek veya kromozom parçaları gibi kromozomal anomaliler elde eğilimli hale bildirmişlerdir. Gerçekten de, enzimatik veya kimyasal disosiasyon 2,3,4 kullanıldığında Pasajlanması için koloniler manuel diseksiyon istikrarlı bir karyotip 5 korur, uzun vadeli kültür sırasında karyotipik değişiklikler gözlenir. Bunun yanı sıra, besleyici hücrelerin ortadan kaldırılması gibi çevre değişiklikleri de hESC 3,6 genetik bütünlüğünü tehlikeye görünüyor. Kromozomal bozukluklar önemli dikkate alınarak hESC embriyogenez çalışmaları ve uyuşturucu testi için gerekli bir araç olarak, in vitro genetik bütünlüğü hESC karakterizasyonu hücresel fizyoloji etkileyebilir. Ayrıca, gelecekte tedavi amaçlı kromozomal değişiklikler sık ​​karsinogenez ilişkili olarak gerçek bir endişe vardır. Burada G-bantlama, FISH, SKY veya CGH teknikleri 7,8 kromozom sonraki analiz için yüksek kalitede kromozom yayılır elde etmek için basit ve kullanışlı bir yöntem gösteriyor . Translokasyonlar ve anöploidiler görünümünü izlemek için rutin kromozomal durumu 5 pasajların aralıklarla kontrol etmenizi öneririz Priscila Britto ve Rafaela Sartore kağıt üzerinde eşit olarak katkıda bulundu.

Watch this Scientific Journal Video about Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping at JoVE.com

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Karyotipleme genetik değişiklikler tespit etmek için yaygın olarak kullanılan basit ve kullanışlı bir bir tekniktir. Burada bakımı, kültür bu hücrelerin kromozom durum izleme için insan embriyonik kök hücrelerin kromozom yayılmış hazırlık için adım protokol tarafından bir adım açıklanmaktadır.

Karyotipik İnsan Embriyonik Kök Hücreleri kromozom Yayılı Hazırlık

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

chromosomal-spread-preparation-human-embryonic-stem-cells-for

Research

JoVE Journal

Biology

48.6K Görüntüleme.  Federal University of Rio De Janeiro-UFRJ. Karyotipleme genetik değişiklikler tespit etmek için yaygın olarak kullanılan basit ve kullanışlı bir bir tekniktir. Burada bakımı, kültür bu hücrelerin kromozom durum izleme için insan embriyonik kök hücrelerin kromozom yayılmış hazırlık için adım protokol tarafından bir adım açıklanmaktadır.

Video: Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Bütünleşmeyen episomal plasmidler kullanılarak dondurulmuş Buffy tüylerdeki uyarılmış pluripotent kök hücrelerin üretilmesi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Gelişim Biyolojisi

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Kromatin fare oosit ilerleme profaz metafaz II analizi için hazırlıklar yaymak

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Genetik

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genom düzenlenmiş İnsan Pluripotent Kök Hücre Hatları oluşturulması: İzolasyon hedefleyen Gönderen

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polikistik Böbrek Hastalığı Olan İnsanlar ve Fareler kromozom anomalileri Eğitim Spektral Karyotipik

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Tıp

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Transforme İnsan Fibroblastlar gelen Proliferatif Tetraploid Hücreleri kurulması

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biyoloji

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Kültür Hücrelerinin itibaren Kromozom Hazırlık

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

İnsan Embriyonik Kök Hücreleri Hedefleme Çinko parmak Nükleazlar Geliştirilmiş Gen

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Fare Spermatocytes segregasyonun kromozom hazırlanması yayılır

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...