GUS GUS-kusursuz bir protokol, Paja Sijacic ve Courtney Hollender paylaşımı için yararlı yorum ve Şekil 1, Detlef Weigel fotoğraflar için: Biz Chunxin Wang pTSO2 inşa için teşekkür ederiz . Z. L s laboratuvarda Araştırma, ABD Ulusal Bilim Vakfı (IOB0616096 ve MCB0744752) tarafından desteklenmektedir. ZL, Maryland Zirai Araştırma İstasyonu Üniversitesi tarafından kısmen destekleniyor.

I. Çiçekli daldırma Arabidopsis thaliana dönüşümü <ol><li> Yaklaşık 1 ay önce dönüşüm, pot başına yaklaşık 10-15 bitkiler ile 4-8 tencere (5 inç kare tencere) üzerine Arabidopsis tohumları ekmek. Plazmid inşa başına 4 tencere bitkilerin normal doğurganlık ile bitkiler için yeterli olacaktır. Doğurganlık azalmış mutant Arabidopsis dönüştürmek için varsa, buna göre kapların sayısını artırmak. </li><li> Bitkiler standart koşullar altında (22, 16 saat ışık ve 8 saat karanlık ° C) yetiştirilmektedir. Kısa gün veya daha düşük sıcaklık altında yetiştirilen bitkiler, bu dönüşüm için hazır hale çiçek ve daha uzun sürecektir. İyi bitki bakımı, başarılı bir dönüşüm için gerekli olan sağlıklı bitkiler, sağlayacaktır. </li><li> Bitkiler sadece cıvatalı ve çiçek başladı. Onlar şimdi, dönüşüm için hazır. Dönüşüm verimliliği artırmak için, dönüşüm 3-4 gün önce, en kısa sürede daha ikincil ateş oluşumunu teşvik etmek cıvatalı gibi ana gelişme sürgünler keserek. Bitkilerin dönüşüm önceki gün Su. Bu toprak, çiçek daldırma sırasında çok infiltrasyon medya emmek olacağını garanti eder. </li><li> : (Agrobacterium tumefaciens gerginlik GV3101 pTSO2 taşıma ile GUS, 50 mcg / ml Kanamisin: pTSO2 durumunda): İki gün önce dönüşüm, uygun antibiyotik içeren 40ml LB aşılamak: GUS inşa. PTSO2: GUS Kanamisin 3 direnç bahşeder NPTII gen sahip pBIN20 vektör klonlanmış. 28-30 ° C&#39;de sallayarak inkübatör gecede büyütün </li><li> Ertesi gün, 50 mg / ml Kanamisin içeren LB 400ml gecelik kültür transferi 8 ml. 28-30 ° C&#39;de sallayarak inkübatör gecede büyütün </li><li> Dönüşüm gününde, OD 600 Agrobacterium kültürünün yaklaşık 0.8 (0.5 1&#39;e arasında OD 600 kabul edilir) ulaştığında, 5000rpm 8 dakika Agrobacterium gecede kültürü aşağı doğru döndürün. </li><li> 1 L infiltrasyon medya Agrobacterium pelet yeniden süspanse (litre) (10mM MgCl 2,% 5 sakkaroz, 0.44mM 6-benzyladenine (BA),% 0.3 silwet L-77, 1x Gamborg vitamin çözüm ve otoklava su). Orta otoklava gereken değildir ama taze yapılmış olması gerekir. 1 L Agrobacterium içeren infiltrasyon orta bir yemek (8 &quot;x15&quot; x2 &quot;Pyrex cam tabak) içine dökün. </li><li> Pot haricindekileri (bitkiler kapalı düşme olmaz) ve bitkilerin tüm hava infiltrasyon çözüm içine daldırma, bir seferde bir tencerede 5 dakika tutun. Bitkinin tüm hava infiltrasyon orta dalmış iken, toprak, toprak üzerinde mantar büyüme şansını azaltmak için herhangi bir infiltrasyon medya içinize izin kaçının. </li><li> Daldırma sonra, bir tepsi kağıt havluyu birkaç kat üzerinde kendi tarafında tüm daldırma tencere yerleştirin. Kağıt havlu infiltrasyon medya erişim miktarı içinize çekin. Tepsi yüksek nem sağlamak için naylon ile örtün. Bitki büyüme odasına tepsiye yerleştirin. </li><li> Ertesi gün, dik plastik wrap ve kağıt havlu, yer ve tencere çıkarın. 4-5 gün daha bu bitkiler su etmeyin. Daha sonra, tohum kadar normalde bitkilerin bakımı ve toplu olarak tohumları toplamak. </li><li> 4 gram serin agar, otoklav ve 1M NaOH ile 5,8, 500 ml su, pH içinde çözülür, MS 50 mg / ml Kanamisin (ağırlık 2.2G Murashige içeren orta ve bazal tuz vitamini olmadan Skoog levhalar (150x15mm petri) dökün ) 50 mg / ml nihai konsantrasyonu Kanamisin ekleyin. Tabaklar gerekli ° C ye kadar 4 tutulur. </li><li> Antibiyotiğe dayanıklı transgenik bitkiler için seçmek için en az 20.000 tohum ekrana ihtiyacı var. 0.1 gram yaklaşık 5000 tohum. Ağırlık ve tohum miktarı tahmin. </li><li> 15 ml Falcon tüp 30 saniye boyunca% 70 etanol ile yıkayın tohum sterilize edin. Tohumları bir kez steril su ile yıkayın. 01:10 seyreltme mağaza satın aldı,% 5 çamaşır suyu (Clorox) içeren bir çözüm için tohum 30 saniye bekletin. Tohumları 3-6 kez steril su ile durulayın. Her MS (Kanamisin) plaka üzerine pipet yaklaşık 5000 tohumlar, steril kaput, Micropore 3M bant ile kirlenmesini önlemek amacıyla plakaları, plaka üzerinde eşit tohum yayıldı. </li><li> 4 plakaları inkübe ° C, 3-4 gün boyunca karanlıkta, sonra bir odaya (aynı bitki büyüme odası olabilir) plakaları aktarmak. Yaklaşık 14 gün sonra, transgenik fide yeşil kalır ama transgenik olmayan soluk dönüm ve ölüyor. Yavaşlama orta kökleri dışarı çekerek ve toprağın yerleştirdiler transgenik fideler toprağa aktarın. </li></ol> II. PTSO2 incelenmesi: GUS ekspresyonu <ol><li> Hasat pTSO2: GUS transgenik fide ve% 90 aseton, bir bardak sintilasyon flakon veya mikrofuge&#39;de tüpleri soğuk koyunbuz üzerinde tutulur. Biri büyük bir sayıda numune analiz eğer Polistiren microtiter plakalar (polipropilen) de kullanılabilir. </li><li> Tüm örnekler hasat sonra, 20 dakika boyunca oda sıcaklığında şişeleri. Bu süre zarfında x-Gluc (% 0.2 Triton X-100, 50mm NaHPO 4 Tampon pH7.2, 2mm Potasyum Ferrocyanide, 2mm Potasyum ferrisiyanür) ve buz üzerinde yer olmadan taze boyama tampon oluşturur. </li><li> Örneklerden aseton çıkarın ve boyama tamponu ekleyin. </li><li> X-Gluc boyama solüsyonu (% 0.2 Triton X-100, 50mm NaHPO 4 Tampon pH7.2, 2mm Potasyum Ferrocyanide, 2mm potasyum ferrisiyanür, 2mm x-gluc) olun. Örneklerinden boyama tamponu çıkarın ve x-Gluc örnekleri içeren boyama tamponu ekleyin. </li><li> 15 ila 20 dakika arasında bir vakum altında örnekleri Infiltrate. Vakum yavaşça serbest bırakın ve tüm örnekleri boyama çözüm yüzeyin altında lavabo doğrulamak. Gerekirse vakum yayımlandıktan sonra, tüm örneklerde lavaboya kadar infiltrasyon tekrarlayın. </li><li> 37 inkübe örnekleri ° C x-Gluc boyama tampon geceleme. </li><li> Inkübatör örnekler çıkarın ve boyama tamponu çıkarın. Her değişiklik için 30 dakika oda sıcaklığında (% 20,% 35 ve% 50 etanol), ardışık etanol serisi örnekleri yıkayın. </li><li> Oda sıcaklığında 30 dakika içinde FAA fiksatif (% 50 Etanol,% 5 Formaldehit,% 10 asetik asit, geri kalanı su) dokuların sabitleyin. FAA çıkarın ve% 70 etanol ekleyin. Bu noktada dokuların görüntülendi olabilir ve bir dijital kamera ile donatılmış bir diseksiyon mikroskobu altında fotoğraflandı. TSO2 (Şekil 1) ifade edilir, koyu mavi renk gösterir . Alternatif olarak, dokuların, 4 ° C daha sonra izlenmek üzere saklanabilir. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Şekil 1" /> Şekil 1 pTSO2:: GUS transgenik fidan ifade (A), yan kök (B), ve kök ucu (C). TSO2 organizatörü, genç ve bölme dokularda son derece aktif olduğunu unutmayın . (C) gösterilen sporadik ifade deseni, belirli bir hücre döngüsü evreleri olarak ifade genlerin tipik bir örneğidir. </li></ol> Temsilcisi Sonuçlar Doğru bir şekilde yapılırsa, dönüşüm verimliliği yaklaşık% 0.1-0.2 olmalıdır. Başka bir kelime, bir, 5000 tohum eleme 5-10 transgenik fide almalısınız. Ortalama, yaklaşık 100 transgenik çizgileri vahşi tip bitkiler 4 tencere dönüştürerek elde edilebilir. Için pTSO2 içeren transgenik fide: GUS inşa 3, GUS faaliyet yansıtan koyu mavi renk, genç yaprak, sürgün apeks, kök ucu ve lateral kök primordia (Şekil 1) de dahil olmak üzere aktif olarak bölünen hücreleri bulunur . Üniform olmayan sporadik desen hücre döngüsü faz-spesifik ifade karakteristik.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

Dönüşüm verimliliği aşağıda tartışılmıştır çok farklı faktörler tarafından belirlenir: <ol><li> Birincil öneme sahip bitkilerin sağlık ve yaşlarına. Üreten çok sayıda olgunlaşmamış çiçek tomurcukları yaklaşık bir aylık bitkiler idealdir. Dönüşüm ikincil ateş oluşumu 3-4 gün önce teşvik etmek birincil sürgünler kapalı Kırpma dönüşüm verimliliği artacaktır. Önceki bitkiler transformants doğuran, ancak daha düşük bir oranda. </li><li> Bitkilerin Doğurganlık ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>6-benzyladenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>852430</td>
<td>&nbsp;</td>
<tr>
<td>Silwet-77</td>
<td>&nbsp;</td>
<td>Crompton Corp.</td>
<td>&nbsp;</td>
<td>Toxic, wear gloves</td>
</tr>
<tr>
<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
<tr>
<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
<td>&nbsp;</td>
<td>Biosynth</td>
<td>B-7300</td>
<td>Toxic, light sensitive keep in the dark</td>
</tr>
<tr>
<td>Micropore 3M Tape</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19027761</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

Bir organizmanın içine yabancı genlerin tanıtmak için yetenek, modern biyoloji ve biyoteknolojinin temelini oluşturur. Model çiçekli bitki<em&gt; Arabidopsis thaliana</em&gt; Çiçek daldırma dönüşüm yöntemi<sup&gt; 1-2</sup&gt; Basitlik, verimlilik ve düşük maliyet nedeniyle önceki tüm yöntemler yerini almıştır. Özellikle genç çiçeklenme, sürgünler<em&gt; Arabidopsis</em&gt; Bitkilerin bir çözüm batırılmış<em&gt; Agrobacterium tumefaciens</em&gt; Belirli plazmid yapıları taşımak. Daldırma sonra, bitkilerin normal büyüme ve verim tohumları küçük bir yüzdesi, yabancı gen ile dönüştürülmüş ve antibiyotik içeren orta için seçilebilir iade edilir. Önemli ölçüde kolaylaştırdı Bu çiçek daldırma yöntemi<em&gt; Arabidopsis</em&gt; Araştırma ve bitki gen fonksiyon anlayışımıza büyük katkıda bulunmuştur. Bu çalışmada, biz, bir muhabir gen dönüştürmek için çiçek daldırma yöntemi kullanın<em&gt; Β-glucuronidase (GUS)</em&gt; Kontrolü altında<em&gt; TSO2</em&gt; Organizatörü.<em&gt; TSO2</em&gt; Ribonukleotid redüktaz (RNR) küçük altbirim için kodlama<sup&gt; 3</sup&gt;, Hücre döngüsü, hücre döngüsünün S-fazında dNDP biyosentezi için gerekli bir düzenlenir gen. İncelenmesi<em&gt; GUS</emTransgenik&gt; ifadesi<em&gt; Arabidopsis</em&gt; Fide olduğunu gösteriyor<em&gt; TSO2</em&gt; Aktif dokularda bölünerek ifade edilir. , Yüksek okul ve üniversite düzeylerinde araştırma için değil, aynı zamanda eğitim için değil, sadece bildirilen deneysel yöntem ve malzemeleri kolaylıkla adapte edilebilir.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

Bu makale, çiçek-daldırma yöntemi göstermektedir.<em&gt; Agrobacterium tumefaciens</em&gt; Aracılı dönüşüm<em&gt; Arabidopsis thaliana</em&gt;. Bir hücre döngüsü düzenlenir organizatörü muhabiri tanıtarak,<em&gt; PTSO2: β-glucuronidase (GUS)</em&gt; Içine<em&gt; Arabidopsis</em&gt;, Bir transgenik fide GUS muhabir ifade nasıl algıladığı göstermektedir.

Floral-daldırma Dönüşüm Arabidopsis thaliana Inceleyin PTSO2: β-glucuronidase Reporter Gen İfadesi

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

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Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

36.1K Görüntüleme.  University of Maryland College Park. Bu makale, çiçek-daldırma yöntemi göstermektedir. Agrobacterium tumefaciens Aracılı dönüşüm Arabidopsis thaliana. Bir hücre döngüsü düzenlenir organizatörü muhabiri tanıtarak, PTSO2: β-glucuronidase (GUS) Içine Arabidopsis, Bir transgenik fide GUS muhabir ifade nasıl algıladığı göstermektedir.

Video: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biyoloji

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

14 gün eski Fideler Dokular protoplast izolasyonu Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Hücre tipi Özgül gösterilmeden FACS yardımlı kullanarak Doku ve Organ spesifik Fitokrom Yanıtları incelenmesi Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Parafin ve Dondurulmuş Bölüm Drosophila Yetişkin Kaslar

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Erken evre Embriyolar verimli ve hızlı İzolasyon<em&gt; Arabidopsis thaliana</em&gt; Tohumlar

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Histokimyasal boyama<em&gt; Arabidopsis thaliana</em&gt; Ortaöğretim Hücre Duvar Elemanları

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Floral-daldırma Dönüşüm Arabidopsis thaliana Inceleyin PTSO2: β-glucuronidase Reporter Gen İfadesi

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