Bu çalışma, NSF hibe İOK # 0615996 tarafından finanse edilmektedir.

Bölüm 1: nematod büyüme ortamının hazırlanması (NGM) Petri kapları C. elegans ve P. Deneysel nematod pacificus genellikle nematodu Büyüme Orta (NGM) 1 içeren 6 cm Petri kapları laboratuvarda kültür. Worms, 20 ° C&#39;de tutulur, ama bir sıcaklık aralığında (4 ila ° C - 25 ° C) inkübe nematod türleri ve deneysel tasarım bağlı. NGM ile plakaları hazırlamak için, standart, steril teknikleri, mantar ve bakteri bulaşmasını önlemek için kullanılması gerekmektedir. NGM plakaları hazırlamak için protokol: <ol><li> 15 g agar, 2.4 g NaCl, 2 g Tripton, 2.72 g KH 2 PO 4, 2 L erlene, su, otoklav ile 1 litre hacim makyaj ve 60 ° C su banyosunda soğutmak için izin tartılır . </li><li> 0.8 ml 1 M CaCl 2, kolesterol (5 mg / ml etanol), ve 1 M MgSO 4. Fungal ve bakteriyel kirlenme, 1 ml streptomisin (100 mg / ml) ve 1ml nistatin (10 mg / ml) önlemek için her bir litre orta eklenebilir. </li><li> Unispense makine kullanma ve steril koşullarda muhafaza medya istenilen plakalar içine dökün. Plakalar yaklaşık 2 / 3 üncü tam dolu olmalıdır. Standart bir 60 mm çapında plaka medya yaklaşık 10 ml gerektirir. Agar tamamen gelified kadar plakaları hareket etmeyin, aksi halde zor stereomikroskopta solucanlar odak agar pürüzlü bir yüzey olacaktır. </li><li> Kuruduktan sonra plakalar hemen 4 numaralı seribaşı veya saklanabilir ° C bakteri ekim kadar. </li></ol> Bölüm 2: OP50 bakteri hazırlanması ve ekim NGM Petri kapları P. pacificus E.coli OP50 gerginlik beslenen laboratuvar ortamında kültüre. OP50 böylece solucanlar 1 görselleştirme kolaylaştıran ince bir çim oluşturan NGM plakalar üzerinde sınırlı bir büyüme vardır. OP50 bir kültür büyümek için, LB-suyu kullanın: <ol><li> 500 ml vidalı kapaklı şişe, 500 ml distile su ve otoklav ile ses makyaj 5 g bactotryptone, 2.5 g maya özü ve 2.5 g NaCl ekleyin. </li><li> Suyu, steril koşullar altında kültür plakasına OP50 tek bir koloni aşılamak ve bu 37 geceleme büyümesine izin ° C </li><li> LB-OP50 kullanılmaya hazır ve kısırlık muhafaza ise 4 ° C&#39;de birkaç ay saklanabilir. Plakalar numaralı seribaşı gerektiğinde, küçük bir steril beher içine küçük bir miktar suyu dökün ve bu stok kültür kirlenmesini önlemeye yardımcı olacaktır. </li><li> Tohum plakaları, 60 mm çap NGM Petri plaka ve yaymak için girdap üzerine OP50 100-120 ul dağıtmak. </li><li> Büyümek için çim için oda sıcaklığında bir gecede bu numaralı seribaşı plakaları inkübe edin. Bu seribaşı plakalar, 3 hafta kadar 4 ° C&#39;de saklanabilir. Ters bir pozisyonda saklanması hızlı kurumasını önlemek yardımcı olur. </li></ol> Bölüm 3: bir seçim yapma Bu bölümde, solucanlar için bir seçim yapmak nasıl açıklar. Almak, solucanlar, tek bir konumdan diğerine aktarmak için kullanılır. Bazı araştırmacılar biraz değişik metal bileşimleri tercih olsa pick 30 göstergesi% 10% 90 platin iridyum tel yapılır: <ol><li> Tel 3-4 cm Kes, Pasteur pipeti (ucu küçük bir uzunluğu kırıldı) ucu ve daha sonra içine 0,5 cm Bunsen beki üzerinden bardağa mühür. Camdan çıkıntılı tel uzunluğu yaklaşık 3-3.5 cm, ancak bireysel tercihlerine göre değişebilir. </li><li> Sonunda, bir çekiç ve pense kullanarak çıkıntılı tel düzleştirin. Sonra bir kepçe formu basık kısmını yukarı doğru bükün. </li><li> Solucan veya agar zarar görmesini engellemek için zımpara kağıdı ile pick keskin kenarları yumuşatmaya. </li></ol> Bölüm 4: Toplama solucanlar <ol><li> , Solucanlar almak bir alev platin tel sterilize ve delinme veya hasar agar yüzeyi bakım alarak, ceket ucu kalın yapışkan bakteri bakteriyel çim boyunca almak basık ucu sürükleyin. </li><li> Stereomikroskopta kullanarak, çok solucan alma bakteri sopalarla kadar alınır solucan / solucanlara karşı yapışkan ucu hafifçe fırçalayın. </li><li> Alma ucundaki solucan sonra, bakteriyel çim karşı yeni plaka almak ucu dokunmadan veya sürgülü hemen yeni plaka transfer ve solucan tarama olmalıdır. Bakım başka solucanlar zor onları almak için deliklere tarama plaka üzerinde agar yüzeyine zarar vermemek için alınmalıdır. Solucan, çok uzun süre pick kalırsa, birbirinden ayrılacak. </li></ol> Bölüm 5: EMS mutagenez Etil metan sülfonat (EMS) 2 genom mutasyonları geniş bir yelpazede neden olur. Mutantlar, yumurtlama, kas defekti gibi kusurları tespit edilebilirlar, cinsiyet tayini, Dauer oluşumu, davranış ve gonad oluşumu. EMS mutagenez için protokol C için açıklanan benzer elegans 1. <ol><li> 1N NaOH 500 ml (veya daha fazla) hazırlayın. </li><li> , Solucanlar tam 6 cm çapında Petri kapları 4-5 atın üçüncü larva jüvenil (J3) aşamada birkaç yüz solucanları içeren ve 2-3 ml steril M9 plaka başına kullanarak kapalı solucanlar yıkayın. </li><li> Steril, tek kullanımlık 15 ml santrifüj tüpüne tüm solucanları toplayın. Solucanlar yayılmak için bir pelet oluşana kadar 5-7 dakika veya 1500 xg&#39;de santrifüjleyin. </li><li> Sıvı aspire ve M9 2 ml yeniden askıya solucanlar kullanabilirsiniz. M9 2 ml içeren bir tüp 20 ul EMS ekleyin ve bunu çözmek için sallayın. Sonra hafifçe solucan süspansiyon ve türbülans 2ml EMS çözüm ekleyin. EMS son konsantrasyonu 47 mM olacak. Dikkat: EMS çok kuvvetli mutajen ve davlumbaz ele alınmalıdır. Bu mutajen ile temas eden tüm malzemeler (eldiven, pipetler, ipuçları) EMS inaktif 1N NaOH ile tedavi edilmelidir. Tüm EMS mutagenez prosedürü sırasında çift eldiven giyin. Kurumun kurallarına göre tehlikeli atık bertaraf edin. </li><li> Santrifüj tüpüne yerleştirin ve parafilm kapağı kapak; davlumbaz yatay olarak yerleştirin ve 3,5 saat süreyle inkübe solucanlar izin. Tüp da düşük bir hızda rocker yerleştirilebilir. </li><li> Tüp parafilm kapağı açın ve kapağı, solucanlar lavaboya kadar dikey konumu ve inkübe yer. </li><li> Süpernatantı aspire ve 1N NaOH içeren bir beher / balona atın. Bu EMS inaktive. </li><li> 5 ml M9 solucanlar, solucanlar bir pelet oluşana kadar 5-7 dakika veya 1500 xg&#39;de solucanlar, santrifüj yıkamak için tüp 25-30 kez ters çevirin. Süpernatantı aspire ve 1N NaOH beher içine atın. </li><li> Çamaşır adım en az 3 kez tekrarlayın. </li><li> M9 az miktarda (~ 500 ul) pelet solucan son yıkamadan sonra, yeniden askıya alma ve OP50 bakteriler içeren bir çim NGM plakalar üzerine pipetle. Bu noktada artık kimyasal kaputu çalışmak gerekiyor. </li><li> Sıvı birkaç dakika kurumasını bekleyin ve sonra taze numaralı seribaşı plakaları almak ve sağlıklı görünen J4 larva transferi. Orijinal plaka kurumun tehlikeli atık kurallarına göre atın. </li><li> Mutagenized solucanlar kendi kendini döller. Bir stereomikroskopta kullanarak, ilgi resesif mutantlar için F2 nesil fenotipleri ekran. </li></ol> Bölüm 6: Psoralen mutagenez Bu bölümde, uzun dalga boyu ultraviyole (UV) ile birlikte TMP kullanarak mutagenez için prosedürü açıklar. Mutagenez TMP / UV yöntemi yaygın bir genom 3 içinde silinmesi mutasyonlar ikna etmek için kullanılır. <ol><li> Önceki yordamda açıklandığı gibi 15 ml santrifüj tüp içine 4-5, 6 cm çapında Petri kapları solucanları toplayın. </li><li> 5 dakika için 1500 xg&#39;de 20 kez ters solucanlar, ve santrifüj yıkamak için, 10 ml M9 ekleyin. Süpernatantı atın ve bir toplam 3 kez yıkama tekrarlayın. Sıvı aspire ve M9 kendi hacmi 30 mcg / ml TMP içeren yaklaşık 10 kez solucanlar yeniden askıya. Pipet 20 M9 2 ml içine TMP ul (3 mg / ml). EMS gibi, TMP güçlü bir kanserojen ve uygun güvenlik önlemleri alarak ele alınması gerekir. UV ışık uygun göz giyer potansiyel risk önlemek için kullanılmalıdır. Tek uygun biyolojik tehlike torbalara atılmalıdır. </li><li> Tüp düşük devirli bir rocker (40 UPM), oda sıcaklığında 15 dakika boyunca karanlıkta inkübe edin. Dikey kapalı tüp solucanlar lavabo izin vermek için 10 dakika olarak ayarlayın. </li><li> Pasteur pipeti ile tüp altındaki solucanlar çıkarın ve seri başı olmayan bir NGM plaka üzerine aktarabilirsiniz. Kurumunuzun tehlikeli kimyasal yönergeleri izleyerek süpernatantı atın. </li><li> Aşırı TMP plaka emilir kadar karanlıkta plaka tutun. </li><li> UV yoğunluk ölçer kullanarak, 500 μW / cm 2 4 plaka üzerinde yoğunluk olduğunu, uzun dalga UV kaynağı ve plaka arasındaki mesafeyi ayarlamak . Alüminyum kapak ve kapak çıkarın ve 50 saniye için uzun dalga UV radyasyonu TMP tedavi solucanları içeren plaka kapak ve maruz. </li><li> Kapak ve oda sıcaklığında 5 saat süreyle karanlıkta solucanlar terk. </li><li> Alış ve taze numaralı seribaşı plakaları sağlıklı görünen J4 larva transferi. Kurumun tehlikeli kimyasal yönergeleri izleyerek orijinal plaka atın. </li><li> Bir stereomikroskopta kullanarak, ilgi mutasyon için F2 nesil fenotipleri ekran. </li></ol> Bölüm 7: Temsilci Sonuçlar Bazı P. mutagenez (ya EMS veya TMP / UV) kaynaklanan pacificus mutant fenotipleri Şekil 1&#39;de gösterilmiştir. Her mutant vahşi tip hayvan kolayca ayırt karakteristik bir fenotip vardır. Solucanlarmutajen, ebeveyn nesil (P0) 30 J3/J4 larvaları ayrı tabak yerleştirilir ve F1 nesil yumurta bırakmak için izin verildi ile tedavi edildi. Sağlıklı F1 döl gösterdi plakaları, toplam 1000 F1s ayrı ayrı kalıplara transfer edildi ve F2 döl fenotipleri gösterildi. Dumpy 5,6, ankoordine 6 ve Trafo 7 fenotipleri Hayvanlar bulundu. <img src="/files/ftp_upload/2293/2293fig1.jpg" alt="Şekil 1" /> Şekil 1 Pristionchus pacificus yabani tip ve mutant hayvanlar. A. wt hermafrodit B. wt erkek C. dpy mutant D. unc mutant E. tra mutant. Top iki panel vahşi tip hermafrodit (solda) ve erkek (sağ) Hayvanların gösteren kalan paneller farklı mutantlar ile karşılaştırmak.

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. 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> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

Mutagenez çeşitli deneysel model organizmaların genetik çalışmalar için yaygın olarak istihdam edilen bir tekniktir. Mutagenez deneyler genellikle bir genin 2 işlevini tanımlamak için kullanılır. Pa sadece burada açıklanan mutagenez prosedürleri kullanılabilir pacificus değil, aynı zamanda C elegans ve ilgili diğer nematod türü. Burada iki yöntem, EMS ve TMP / UV mutagenez açıklar. EMS genom 3,8 boyunca nokta mutasyonl...

an introduction to worm lab: from culturing worms to mutagenesis

Ultraviyole ışığı ile birlikte (TMP / UV) etil metan sülfonat (EMS) ve 4, 5 &#39;, 8-Trimethylpsoralen: Bu protokol, laboratuvar ve nasıl iki alternatif yöntemler kullanarak onları mutagenize nematod korumak için prosedürler açıklanmaktadır. Nematodlar, çünkü kendinden gübreleme hermafroditlerde ve hayvan başına ahfadının yüzlerce üretebilir erkek oluşmaktadır basit vücut planı ve çiftleşme sistemi, genetik çalışmalar için güçlü bir biyolojik sistemler. Nematod, bakteri çim içeren agar plaklarına korunur ve kolay bir plakadan bir pick kullanarak başka bir transfer edilebilir. TMP / UV esas silmeler indükler EMS, nokta mutasyonları ve küçük delesyonlar ikna etmek için yaygın olarak kullanılan bir alkilleyici ajandır. Nematod kullanılan türlerin bağlı olarak, EMS ve TMP konsantrasyonları optimize edilmesi gerekecektir. Nematod Pristionchus pacificus resesif mutasyonlar izole etmek için, F2 nesil hayvanlar görsel fenotipleri için tarandı. Bu yöntemleri göstermek için, solucanlar mutagenized ve koordinasyonsuz (UNC), Dumpy (dpy) ve Trafo (Tra) mutantlar aradı.

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Tarama fenotipik defekti olan mutantlar için, belirli bir biyolojik süreç fonksiyonu genleri tanımlamak için basit bir yöntem. Bu yazıda, kültür serbest yaşayan solucanlar (örneğin, nasıl tarif<em&gt; Pristionchus pacificus</em&gt;) Laboratuvar ve iki farklı mutagenez yöntemleri, EMS ve TMP / UV göstermektedir.

Solucan Lab An Introduction to: Kültürleme Worms Mutagenez

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

an-introduction-to-worm-lab-from-culturing-worms-to-mutagenesis

Research

JoVE Journal

Biology

34.3K Görüntüleme.  University of Texas at Arlington. Tarama fenotipik defekti olan mutantlar için, belirli bir biyolojik süreç fonksiyonu genleri tanımlamak için basit bir yöntem. Bu yazıda, kültür serbest yaşayan solucanlar (örneğin, nasıl tarif Pristionchus pacificus) Laboratuvar ve iki farklı mutagenez yöntemleri, EMS ve TMP / UV göstermektedir.

Video: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Tüm Plazmidler Homemade Sitesi Yönetmen Mutagenez

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biyoloji

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Tek Oosit Bisülfit Mutagenez

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Elektrofizyoloji Tabanlı Katı Desteklenen Membran Giriş

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Bir In vivo</emŞu Protein Kompleksler yalıtmak için> Çapraz yaklaşım Drosophila Embriyolar

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Kültürleme<em&gt; Caenorhabditis elegans</em &gt; Mikroparçacık bombardımanı ile aksenik Sıvı Medya ve Transgenik Worms Yaratılış

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Çekirdek Biyoloji Kavramları öğretmek için Fare Meme Tümör Hücreleri kullanma: Basit Lab Modülü

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Laktaz enzim etkinliğin ölçülmesi öğretim laboratuarında

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Manyetik aktif hücre stratejileri yalıtmak ve Sinovyal sıvı elde edilen mezenkimal kök hücre bir tavşan modelinden arındırmak için sıralama

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...