Name: measuring gene expression in bombarded barley aleurone layers with increased throughput
Uploaded: 2018-03-30T13:30:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Yazarlar Greyson Butler ve Margaret Barrett deneyler, tahıl homojenizasyon tavsiye için Judy taş ve Lynn Hannum tavsiye fluorometry için yürütmek yardım için teşekkür ederiz. Bu eser Ulusal Bilim Vakfı tarafından (IOB 0443676), bir kurumsal geliştirme Ödülü (fikir) üzerinden Ulusal Genel tıbbi Bilimler Enstitüsü Ulusal Sağlık Enstitüleri grant numarası P20GM0103423 altında ve gelen hibe tarafından desteklenmiştir Doğa Bilimleri Colby üniversite bölümü.

1. hazırlanması efektör ve muhabir gen yapıları
<ol><li>Kurucu organizatörü (örneğin Mısır Ubikuitin, UBI) istihdam efektör yapıları için sürücü ifade istenen açık okuma çerçevesi oluşturun. Örneğin, sürücü güçlü kurucu ifade TaABF15 UBI::TaABF1 içeren bir plazmid oluşturmak.</li>
	<li>Olan faaliyet ölçülebilir olmaktır, organizatörü içerir muhabir yapıları GUSgibi bir açık okuma çerçevesi, ters yönde yerleştirilir kurmak. Örneğin, ifade HVA1 organizatörü2,5ölçmek için HVA1::GUS içeren bir plazmid oluşturmak.</li>
	<li>Bir iç kontrol inşa inşa (örneğin UBI::luciferase).</li>
	<li>Silis bağlama sütun kullanarak ( Tablo malzemelerigörmek) protokol, yüksek kaliteli plazmid gerekli gen yapıları, her biri için hazırlamak. Morötesi Spektroskopi kullanarak ( Tablo malzemeleri, dikkatle bakınız) her plazmid hazırlık konsantrasyonu ölçmek.</li>
</ol>2. bombardıman için arpa taneleri hazırlanması
<ol><li>Bir e-tablo ayarlamak (bir örnek için bkz: şekil 1 ) bombardıman edilecektir plazmid, uygun hormon tedavileri ve deneyin bir parçası olacak her tedavi için diğer ilgili bilgileri gösteren.</li>
	<li>Bir kesme tahtası ve steril bir jilet kullanarak, tahıl Himalaya arpa (40 denemeye dahil her tedavi için) gelen embriyo kesti. Renksiz ya da ortalamadan daha önemli ölçüde daha küçük taneler hariç.</li>
	<li>Embryoless tahıl 50 mL tüp içine yerleştirin. Su ve rock 40 mL 10 dakika için ekleyin.</li>
	<li>40 mL % 10 çamaşır suyu ekleyin ve dikkatle (tahıl kaybetmemek için) suyu dökün. Kaya 20 dakikadır.</li>
	<li>Çamaşır suyu dökmek ve 40 mL steril su ekleyin. Rock 5 dakika. Bu steril su yıkama dört kez daha tekrarlayın.</li>
	<li>40 mL Imbibing çözeltisi (20 mM sodyum süksinat, 20 mM Kalsiyum klorür, pH 5,0) ekleyin ve 40 μL kloramfenikol hisse senedi çözüm (10 mg/mL).</li>
	<li>Imbibing Çözümle (kloramfenikol) çoğunu tüp sizden bir vermikülit tabak aktarmak ( Tablo malzemelerigörmek). Ardından taneleri ıslak filtre kağıdı vermikülit plaka yüzeyi üzerine aktarın. Tahıl eşit yayılmış. Pour kısmen (ama tamamen değil) olduğu tohumlar tutmak için yeterli imbibing çözümde sular altında. Vermikülit plaka üzerine kapak koyun.</li>
	<li>48 h ile floresan lambalı aydınlatma için 24 ° C'de embryoless tahıl kuluçkaya. Ek Imbibing çözüm gerektiği gibi ekleyin.</li>
	<li>90 mm plastik petri kabına kadar açın ve çözüm Imbibing 20 mL çanak içine kendisi ve 20 mL ters kapağı dökün. Kloramfenikol (10 mg/mL) 20 μL her ekleyin. İyi noktası forseps iki çift alkol daldırma tarafından sterilize.</li>
	<li>Birkaç embryoless tahıl petri kabına kapağı yerleştirin. Forseps kullanarak, her tahıl (saydam) tohum kat yavaşça çıkarın. Tohum kat tahıl pürüzsüz kısmından kaldırılırken, altındaki (aleurone) hücreler zarar vermeyen. Soyulmuş tahıl Imbibing solüsyon içeren petri kabına aktarın.</li>
	<li>Tohum kat tüm taneleri soyma sonra onları vermikülit plakasına geri. Soyulmuş embryoless tahıl 24 ° c 16-20 saat boyunca kuluçkaya.</li>
	<li>Sadece tahıl bombardıman için kullanmadan önce yüzey nemi petri kabına gazetelerde filtre arasında sallayarak kaldırın.</li>
</ol>3. bombardıman için plazmid DNA hazırlanması
<ol><li>1.5 mL tüp bombardıman denemeye dahil edilecek her tedavi için etiket. Her tüpün UBI2,5 µg ekle::luciferase iç kontrol plazmid, 2,5 µg GUS muhabir Plazmid ve bir efektör plazmid istenilen miktarda (genellikle 0.025 µg 2,5 µg için). Toplam hacim için 5 µL getirmek için su ekleyin.</li>
	<li>(Bkz: Malzemeler tablo) 1.6 µm altın microcarriers hazırlamak aşağıdaki protokolleri6yayınlandı.</li>
	<li>Her tedavi için askıya alınmış microcarriers 50 µL plazmid DNA içeren 1,5 mL tüp içine ekleyin. Plazmid DNA ile microcarriers göre üreticinin iletişim kuralları6kat.</li>
	<li>Plazmid kaplı microcarriers 48 µL % 100 etanol, damlalıklı 8 μL her beş macrocarriers askıya alınmış microcarriers, askıya alınır ve hava için izin zaman son adımda, Kuru.</li>
</ol>4. partikül bombardımanı Embryoless arpa taneleri
<ol><li>(Bkz: Malzemeler tablo) partikül bombardımanı aparatı ayarla6.</li>
	<li>1550 psi rüptürü disk istinat kap yerleştirin ve araç üzerinde monte edin.</li>
	<li>(Birlikte bir durdurma ekran) istenen plazmid kombinasyonu içeren bir macrocarrier microcarrier denize indirmek derleme yerleştirin. Derleme bombardımanı odası en iyi yuvasına takın. Kap ve macrocarrier kapak kapak standart uzaklık (6 mm) istinat rüptürü disk arasındaki mesafeyi ayarlamak ve durdurma ekran desteği sağlayan bir macrocarrier seyahat etmek mesafe 11 mm (şekil 2A) (Standart) orta konumda yer.</li>
	<li>8 embryoless tahıl sıkı bir Radyal desen aşağı düz'e 90 mm petri kabına kapağı bantlanmış bir filtre kağıdı daire (içe dönük ince uçları) ile düzenlemek (bkz. şekil 2B).</li>
	<li>Durdurma ekranından 6 cm mesafeden bombardımanı odası taneleri ile çanak yerleştirin.</li>
	<li>Bombardıman odası için 95 kPa tahliye ve örnek ile bombardıman.</li>
	<li>Vakum bırakmadan sonra alı tahıl 4 mL Imbibing 1 mg/ml kloramfenikol içeren çözeltisi içeren bir etiketli 60 mm petri kabına aktarın.</li>
	<li>Tüm bombardımanları tamamlayıncaya kadar yineleyin. (Bir platformda sallayarak 100 rpm) 24 ° C'de 24 saat boyunca kuluçkaya.</li>
</ol>5. çıkarma çözünür protein alı tahıl
<ol><li>Forseps kullanarak, 60 mm petri kabına 8 alı tahıl kaldırmak ve onları bir kağıt havlu üzerinde leke. Taneleri her 800 μL içeren iki etiketli 2.0 mL tüpler (tüp başına 4 tahıl) içine bölmek taşlama arabellek (100 mM Na fosfat pH 7.2, 5 mM DTT, 10 µg/mL leupeptin, % 20 gliserol) ve 5 mm paslanmaz çelik boncuk. Tüm alı tahıl için bu adımları tekrarlayın.</li>
	<li>(48) 2.0 mL tüpler boncuk homogenizer iki rafları yerleştirin (bkz. Tablo malzeme). Raflar üzerinde homogenizer bağlayın.</li>
	<li>Örnekleri 30 Hz'de 3 dakika çalkalanır.</li>
	<li>Homogenizer raflar homogenizer kaldırmak ve yeniden örnekleri soğuması için buza (5 dk) koyun.</li>
	<li>Rafları geri homogenizer - ters yönde bağlama. Örnekleri daha 30 Hz'de 3 dakika çalkalanır.</li>
	<li>Raflar on Ice, serin o tekrar adımları 5.3 5.5 için. Bu sallayarak 12 dakika toplam için eklemek olacaktır.</li>
	<li>16.000 x g 4 ° c de tahıl özler 10 dakika santrifüj kapasitesi.</li>
	<li>
Hemen sonra Santrifüjü dikkatle boşaltmak açık supernatants microcentrifuge tüpler ikinci bir dizi içine. Her tüpün hızlı bir girdap verin. Tüpler buza saklayın.</li>
	<li>Bu yüzden onlar yıkanmış ve yeniden süpernatant aktardıktan sonra çelik boncuk çökeltilerini kurtarmak.</li>
</ol>6. ölçüm tahıl özler Luciferase etkinliği
<ol><li>12 x 75 mm cam 200 μL Luciferase tahlil karışımı içeren tüp bebek denetlesinler için tahıl özü her tüp için hazırlamak (45 mM Tris pH 7,7, 15 mM MgCl2, 20 mM DTT, 1.5 mM EDTA, 1 mM biyoluminesans, 1 mM ATP sülfat).</li>
	<li>İlk tahıl ekstresinin 100 μL ilk tahlil tüp ekleyin. Girdap hızla karıştırın. Hemen tüp luminometer tüp yuvasına yerleştirin ve kapıyı kapat. Ölçüm sona erdiğinde, tüp kapıyı sonraki tüp yerleştirilmesi için açık bırakarak luminometer – kaldırın.</li>
	<li>Tüm örnekleri ölçülen kadar bu işlemi yineleyin.</li>
</ol>7. ölçüm tahıl GUS etkinliği ayıklar
<ol><li>GUS tahlil arabelleği (50 mM Na fosfat pH 7.2, 2.5 mM 4-methylumbelliferyl - D-glucuronide, 10 mM DTT, 2 mM EDTA, 10 µg/mL leupeptin, % 20 metanol, % 0.02 sodyum azid) 200 μL 96 iyi plaka her şey için ekleyin.</li>
	<li>Yer 50 μL her tahıl özü de buna karşılık gelen içine. Ucu ile ekledikten sonra karıştırın. Üst film mühürleme ile kapatın.</li>
	<li>96 iyi plaka karanlıkta 37 ° C'de 20 saat boyunca kuluçkaya.</li>
	<li>96 iyi plaka 4.000 x g 4 ° c de 10 dakika santrifüj kapasitesi ve buz üzerinde yerleştirin.</li>
	<li>250 μL 0.2 M Na2CO3 siyah 96 iyi plaka her şey için ekleyin.</li>
	<li>Her centrifuged GUS tahlil karışımı 6.25 μL karşılık gelen içine eklemek iyi (içeren 0.2 M Na2CO3) siyah 96 de plaka. Ucu ile ekledikten sonra karıştırın. Wells ile 10 mikron, 40 mikron ve 100 mikron 4-methylumbelliferone 6.25 μL standartları olarak içerir.</li>
	<li>Siyah plaka plaka yer yerleştirin ve methylumbelliferone floresan okuyun. 360 bir uyarma dalga boyu okumalar alıp nm ve emisyon dalga boyu 460 nm. Verir bir okuma 1000 Floresans birimlerinin 40 mikron 4-methylumbelliferone bir de içeren 6.25 μL ve 250 μL 0.2 M Na2CO3 kazanç enstrümanın ayarlayın.</li>
</ol>8. veri analizi
<ol><li>Değerleri luciferase ve GUS deneyleri için bir elektronik tabloya girin. Bu gen yapıları başarıyla aleurone hücrelere teslim değil anlamına gelir olarak dikkate herhangi bir örnek bir luciferase okuma ile 20.000 göreli ışıldama birimleri s-1, daha düşük hariç.</li>
	<li>(Dört embryoless tahıl grubu temsil eden) için başarıyla alı her tahıl özü, ham GUS ve luciferase veri normalleştirilmiş GUS ifadeden aşağıdaki gibi hesaplar: GUS normalleştirilmiş = [(GUS ifade - GUS boş) (2.000.000) x] / (luciferase ifade-luciferase boş). Not: Bu ne 2.000.000 bir luciferase okuma verdi bir bombardıman gözlenen için GUS etkinliğinin miktarı normalleştirir.</li>
	<li>Bir özel muhabir yapı efektör yapıları veya effectors veya hormon yokluğunda ifade seviyeye karşılaştırarak test edilen hormon tedavileri huzurunda ifade göreli düzeyini rapor.</li>
</ol>

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Yazarlar ifşa gerek yok.

Efektör gen getirilmesi yolu ile partikül bombardımanı oluşturur, geçici muhabir yapıları, deyimden GA/ABA sinyal ve ortaya çıkan hormon düzenlenir gen belirli aktörlerin rolü dissekan için değerli bir stratejidir ifade. Ancak, bu tür deneyleri arpa aleurone hücreleri2,3,4,5,6 taşımak için varolan çok emek yoğun iletişim kurallarıdı...

Arpa aleurone katman hormon düzenlenir gen ekspresyonu bitkiler1incelenmesi için köklü modeli sistemidir. Özellikle, stres yanıt ile ilişkili genlerin absisik asit (ABA) tarafından aktif hale gelir iken çimlenme veya erken fide gelişimi için gerekli genler bir dizi Giberellin (GA), tarafından etkinleştirilir. GA-aktive bazı genlerin ifade ABA ve ahlak bozukluğu-çok yönlü1tarafından inhibe gibi GA ve yolları sinyal ABA, iç içe geçmiş bulunmaktadır.
GA/ABA sinyal içinde belirli aktörlerin rolü efektör gen yapıları partikül bombardımanı, üzerinde oluşturduğu efekt izin muhabir yapıları, geçici ifade tarafından takip yoluyla getirilmesi olmuştur anlamak için değerli bir strateji aşağı akım gen ekspresyonu belirlenecek. Muhabir gen GUS (β-glucuronidase) veya Luciferase gibi özellikle efektör yapı almış hücrelerde gen ekspresyonu hassas ve nicel ölçüm sağlar. Örneğin, ABA kaynaklı genlerin HVA1 gibi Amy32b gibi GA kaynaklı genlerin ise TaABF1 tarafından indüklenir kodlama transkripsiyon faktörü TaABF15,6 bir efektör yapı getirilmesi kurulan baskı. Partikül bombardımanı deneysel bir strateji olarak GA/ABA sinyal çeşitli yönlerini incelemek için birden fazla Laboratuvarları tarafından kullanılmıştır. Bu iş yol organizatörü öğeleri2 GA kaynaklı ve ABA kaynaklı genlerin3aktivasyonu için önemli tanımlaması ve protein kinaz4 keşfi ve transkripsiyon faktörleri düzenleyen5 Bu genlerin ifade.
Varolan iletişim kuralları2,3,4,5,6 partikül bombardımanı ve geçici gen ekspresyonu sonraki ölçümü için oldukça emek yoğun, her kümesi, bombardıman olarak vardır güç bela taneleri bir harç ve havan Tokmağı elle homojenize ve enzim deneyleri ayrı ayrı yapılmaktadır. Bu el yazması kısmen otomatikleştirir ve homojenizasyon adım akıcılık, gelişmiş bir iletişim kuralını raporları ve GUS anlamlı olarak daha fazla işlem hacmi, izin vermek için aynı deneyde, test edilecek tedaviler daha çok sayıda izin deneyleri ve/veya Her tedavi daha istatistiksel olarak sağlam sonuçlar elde etmek için daha fazla çoğaltır eklenmesi. Temsilcisi sonuçları muhabir yapıları, HVA1 ve Amy32b de GA, ABA ve diğer düzenleyici molekül transkripsiyon faktörü TaABF1 tarafından düzenlenir, ifade için gösterilir.

<table><tbody><tr><td>GeneElute HP plasmid Maxiprep kit</td><td>Sigma</td><td>NA0310-1KT</td><td></td></tr><tr><td>UV-vis spectrophotometer</td><td>Nanodrop</td><td>ND-1000</td><td></td></tr><tr><td>Himalaya barley grains</td><td>/</td><td>/</td><td>A variety of hulless barley (store in the dark at 4&amp;deg; C)</td></tr><tr><td>sodium succinate</td><td>Sigma</td><td>S2378</td><td>Reagent for Imbibing Solution</td></tr><tr><td>calcium chloride (dihydrate)</td><td>Fisher</td><td>C79-500</td><td>Reagent for Imbibing Solution</td></tr><tr><td>Imbibing Solution</td><td>home made</td><td>/</td><td>20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.</td></tr><tr><td>chloramphenicol</td><td>Sigma</td><td>C0378</td><td>Prepare a 10 mg/mL stock solution in 70% ethanol.</td></tr><tr><td>vermiculite</td><td>Fisher</td><td>NC0430369</td><td>Used for vermiculite plates.</td></tr><tr><td>filter paper circles (90 mm)</td><td>Whatman</td><td>1001 090</td><td>Used for vermiculite and for pre-bombardment grain preparation</td></tr><tr><td>Vermiculite Plates</td><td>home made</td><td>/</td><td>Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.</td></tr><tr><td>forceps (fine pointed)</td><td>Fisher</td><td>13-812-42</td><td>Used for removing seed coat from barley grains.</td></tr><tr><td>forceps (ultra fine point)</td><td>Fisher</td><td>12-000-122</td><td>Used for removing seed coat from barley grains.</td></tr><tr><td>gold microcarriers (1.6 &amp;mu;m)</td><td>BioRad</td><td>1652264</td><td></td></tr><tr><td>macrocarriers</td><td>BioRad</td><td>1652335</td><td></td></tr><tr><td>calcium chloride (dihydrate)</td><td>Fisher</td><td>C79-500</td><td>Prepare a 2.5 M stock solution and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>spermidine</td><td>Sigma</td><td>S0266</td><td>Prepare a 100 mM stock solution and store 500 &amp;mu;L aliquots at -20&amp;deg; C (use within 2 months).</td></tr><tr><td>rupture discs (1550 psi)</td><td>BioRad</td><td>1652331</td><td></td></tr><tr><td>stopping screens</td><td>BioRad</td><td>1652336</td><td></td></tr><tr><td>macrocarrier holders</td><td>BioRad</td><td>1652322</td><td></td></tr><tr><td>Biolistic particle delivery system</td><td>BioRad</td><td>PDS-1000/He</td><td></td></tr><tr><td>sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td>Reagent for 1M sodium phosphate pH 7.2</td></tr><tr><td>sodium phosphate dibasic</td><td>Sigma</td><td>S9763</td><td>Reagent for 1M sodium phosphate pH 7.2</td></tr><tr><td>1M sodium phosphate pH 7.2</td><td>home made</td><td>/</td><td>Combine 6.9 g of sodium phosphate monobasic monohydrate with 7.1 g of sodium phosphate dibasic. Add water to 100 mL. Add NaOH to get pH 7.2.</td></tr><tr><td>dithiothrietol (DTT)</td><td>Sigma</td><td>43819</td><td>Dissolve in water to 1 M. Store at -20&amp;deg; in 1 mL aliquots.</td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L2884</td><td>Dissolve in water to 10 mg/mL. Store at -20&amp;deg; C.</td></tr><tr><td>glycerol</td><td>Sigma</td><td>G5516</td><td>Prepare a 50% solution in water.</td></tr><tr><td>Grinding Buffer</td><td>home made</td><td>/</td><td>Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 &amp;mu;L of 1 M DTT, 100 &amp;mu;L of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.</td></tr><tr><td>stainlesss steel beads (5 mm)</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>2.0 mL tubes</td><td>Eppendorf</td><td>22363352</td><td>This specific model of tube is recommended for use with the homogenizer.</td></tr><tr><td>bead homogenizer (TissueLyser)</td><td>Qiagen</td><td>85210</td><td></td></tr><tr><td>12mm x 75 mm glass test tubes</td><td>Fisher</td><td></td><td></td></tr><tr><td>luciferin</td><td>Goldbio</td><td>LUCK-100</td><td>Prepare a 25 mM stock solution and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>ATP</td><td>Sigma</td><td>A7699</td><td>Prepare a 100 mM stock solution and store 250 &amp;mu;L aliquots at -20&amp;deg;&amp;nbsp;C.</td></tr><tr><td>Tris base</td><td>Sigma</td><td>T1503</td><td>Reagent for 1M Tris sulfate pH 7.7.</td></tr><tr><td>sulfuric acid</td><td>Sigma</td><td>258105</td><td>Reagent for 1M Tris sulfate pH 7.7.</td></tr><tr><td>1M Tris sulfate pH 7.7</td><td>home made</td><td>/</td><td>Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.</td></tr><tr><td>magnesium chloride</td><td>Sigma</td><td>M9397</td><td>Dissolve in water to 2 M.</td></tr><tr><td>Luciferase Assay Buffer (LAB)</td><td>home made</td><td>/</td><td>Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 &amp;mu;L of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 &amp;mu;L of 0.5 M EDTA. Add water to 50&amp;nbsp;mL.</td></tr><tr><td>Luciferase Assay Mixture</td><td>home made</td><td>/</td><td>Combine 15 mL of LAB, 800 &amp;mu;L of 25 mM luciferin, 200 &amp;mu;L of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20&amp;nbsp;mL) for 100 luciferase assays.</td></tr><tr><td>luminometer (Sirius)</td><td>Berthold</td><td>/</td><td></td></tr><tr><td>4-methylumbelliferyl-&amp;beta;-D-glucuronide (MUG)</td><td>Goldbio</td><td>MUG1</td><td>Dissolve in DMSO to 100 mM.</td></tr><tr><td>sodium azide</td><td>Sigma</td><td>S8032</td><td>Prepare a 2% stock solution in water and store 1 mL aliquots at -20&amp;deg; C.</td></tr><tr><td>96 well plates (standard)</td><td>Fisher</td><td>12565501</td><td></td></tr><tr><td>GUS assay buffer</td><td>home made</td><td>/</td><td>Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 &amp;mu;L of 0.5 M EDTA, 1 mL of 1 M DTT, 100 &amp;mu;L of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.</td></tr><tr><td>TempPlate sealing film</td><td>USA Scientific</td><td>2921-1000</td><td></td></tr><tr><td>96 well plates (black)</td><td>Costar</td><td>3916</td><td></td></tr><tr><td>sodium carbonate</td><td>Sigma</td><td>S7795</td><td>Prepare a 200 mM solution in water.</td></tr><tr><td>4-methylumbelliferone</td><td>Sigma</td><td>M1381</td><td>Prepare a 100 &amp;mu;M solution in water. Freeze 1 mL aliquots at -20&amp;deg;&amp;nbsp;C.</td></tr><tr><td>microplate fluouresence reader</td><td>Bio-Tek</td><td>FLX-800</td><td></td></tr></tbody></table>

measuring gene expression in bombarded barley aleurone layers with increased throughput

Arpa taneleri aleurone tabakası bir hormon düzenlenir gen ekspresyonu tesislerinde önemli model sistemidir. Aleurone hücrelerdeki genler çimlenmesi için gerekli ya da stres yanıt ile ilişkili genlerin absisik asit (ABA) tarafından aktif hale gelir iken erken fide geliştirme Giberellin (GA), tarafından aktive. GA ve ABA sinyal verme mekanizmaları muhabir gen yapıları partikül bombardımanı, via aleurone hücreye enzim deneyleri kullanarak ölçülen elde edilen geçici ifadesi ile tanıtımı tarafından sorguya çekilmeyi. Gelişmiş bir iletişim kuralı bu kısmen otomatikleştirir ve tahıl homojenizasyon adım ve enzim deneyleri düzene bildirilmektedir varolan yöntemlerine göre önemli ölçüde daha fazla üretilen iş sağlar. Homojenizasyon tahıl örneklerinden bir otomatik doku homogenizer kullanarak gerçekleştirilir ve GUS (β-glucuronidase) deneyleri bir 96-şey plaka sistemi kullanılarak yapılmaktadır. Temsilcisi sonuçları iletişim kuralını kullanarak fosfolifaz D faaliyet HVA1 gen ekspresyonu ABA tarafından transkripsiyon faktörü TaABF1 aracılığıyla etkinleştirme önemli bir rol oynamaktadır öneririz.

Burada açıklanan teknik herhangi bir test gen yapısı arpa taneleri aleurone hücrelere tanıtmak için kullanılabilir. Test gen ifade düzeyini daha sonra olabilir uygun ölçülen (Şekil 2). Burada büyük ölçüde açıklanan yüksek üretilen iş Protokolü tohum öğütme ve enzim tahlil adımları verimliliğini artırır. Bu yöntem transkripsiyon faktörü TaABF15,6 ifade ABA kaynaklı g...

Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Gelişmiş bir iletişim kuralı muhabir yapıları arpa aleurone hücrelerdeki geçici gen ifadeden sonra partikül bombardımanı ölçümü için sunulmaktadır. Otomatik tahıl 96-şey plaka enzim deneyleri ile bileme yordamı için yüksek üretilen iş sağlar.

Gen ifadesinin artan işlem hacmi ile alı arpa Aleurone katmanlarında ölçme

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

measuring-gene-expression-bombarded-barley-aleurone-layers-with

Universidad San Sebastian

Research

JoVE Journal

Genetics

6.4K Görüntüleme.  Colby College. Gelişmiş bir iletişim kuralı muhabir yapıları arpa aleurone hücrelerdeki geçici gen ifadeden sonra partikül bombardımanı ölçümü için sunulmaktadır. Otomatik tahıl 96-şey plaka enzim deneyleri ile bileme yordamı için yüksek üretilen iş sağlar.

Video: Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Maya S. cerevisiae hipoksik yanıt sırasında zamanla mRNA düzeyleri ölçme

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Genetik

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolik 4-thiouracil ve bir miktar ile etiketleme yeni mRNA RNA polimeraz II etkinlik için bir Proxy olarak sentezlenmiş

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Isolation of High Purity Tissues from Developing Barley Seeds

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.

Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Gen Ekspresyonu Profilleme için Modifiye Yöntemi ile Tahıl Tohumlarından Yüksek Kaliteli Transkripsiyon Verilerinin Elde Edilmesi

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Biyokimya

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Zebra balığı Oligonükleotid Mikroarray Platformu kullanarak Global Gen İfadesi Analizi

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Biyoloji

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

-Cell Hücre makromoleküler Ulaştırma Testi Planta Biyolistik Bombardıman kullanımı

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

Damlacık Tabanlı RNA Sıralamaile Yüksek EldeLi Maya Zorlanma Fenotilik

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Biyomühendislik

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

Tek Maya Hücreleri MAPK İsteyerek İfade zamansal Kantitasyonu

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...