Cairn araştırma özellikle Mark Henson, Jeremy Graham ve Joao Correia bu sistemi geliştirmek için bizimle çalışmak için teşekkür etmek istiyorum. Biz de David Welsh ve bayram Reddy Peter Laskey (eski adı, Hamamatsu) yanı sıra tasarım aşaması sırasında değerli tartışma için kritik onun girişi el yazması için bir demo kamera ve David Wong kredi düzenlenmesi için teşekkür ederim.

<ol>
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A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+mammalian+body+temperature+can+sustain+peripheral+circadian+clocks.">Rhythms of mammalian body temperature can sustain peripheral circadian clocks.</a> Current Biology. 12 (18), 1574-1583 (2002).</li><li>Balsalobre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+of+circadian+time+in+peripheral+tissues+by+glucocorticoid+signaling.">Resetting of circadian time in peripheral tissues by glucocorticoid signaling.</a> Science. 289 (5488), 2344-2347 (2000).</li><li>Balsalobre, A., Marcacci, L., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+elicit+circadian+gene+expression+in+cultured+Rat-1+fibroblasts.">Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.</a> Curr. 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Hiçbir çıkar çatışması adlı biri yok.

Burada açıklanan memeli hücre kültürü, derin ve statik koşullarda her ikisi de iletişim kuralıdır. Ancak, timsah kolayca diğer sistemleri modellemek için adapte edilebilir. Gerçekten, o zaten aynı anda hareket, uyku ve sürekli karanlık15altında. tutulan Drosophila melanogaster periferik gen ifade ritimler izlemek için mükemmel bir platform sağlamaktadır gösterilmiştir Ayrıca uygulamaya bağlı olarak, burada açıklanan dışındaki kamera türleri uygun olabileceğini kaydetti. Biz uygun filtreleriyle, geçerli ayarı değiştirilmiş bir sürümü prensipte Floresans miktar için kullanılan olabileceğini öngörmektedir.
Özellikle yüksek Uzaysal çözünürlük görüntüleme memeli dilim organotypic PER2::LUC ifadede kronolojik zamanmekansal organizasyon gibi gerekli olduğu için timsah uygun olmayabilir uygulamalar yalnızca bunlar suprachiasmatic çekirdeği, ya da diğer küçük dokusu dilimlerin.
Timsah şimdiye kadar geleneksel kayıt teknikleri tarafından kolayca ulaşılabilir olmamıştır gerçekleştirilecek birçok deney sağlar. Bioluminescence ölçümü için geçerli yöntemleri ile karşılaştırıldığında, timsah her iki tip hücre kültür çanak veya kullanılabilir slayt, dış ortam koşulları, duyarlılık ve processivity düzeyde esneklik sağlar.
Bu özellikle ilgili bir anda ne zaman standart 2D hücre kültür modelleri 3D organoid ve akış kültür sistemleri doğru uzak dur. Bu nedenle, timsah bioluminescence hangi birçok gün ve hafta çok çeşitli koşullar altında üzerinde ölçülebilir uyarlanabilir bir yöntem sağlayacaktır tahmin edilmektedir.

Luciferases gazetecilere gen ekspresyonu ve protein etkinliği olarak kullanımı moleküler ve hücre biyolojisi araştırmalarında popüler bir teknik haline gelmiştir. Bu ateş böceği luciferase sentezi ve katalitik inactivation Kinetik nerede üzerinden yaklaşık 24 s sirkadiyen döngüsü meydana gen ekspresyonu boyuna değişimler raporlama için özellikle uygundur sirkadiyen alanında geçerlidir. Bu nedenle, luciferase Mantarlar, bitkiler, sinekler ve memeliler1,2,3,4da dahil geniş bir aralığında sirkadiyen muhabir olarak istihdam edilmektedir.
Sirkadiyen gen ifade vitromiktarının, bir photomultiplier tüp (PMT) yaygın olarak kollarındaki sinyal kaydetmek için kullanılır. Devresel_ödeme tabanlı ölçümleri esneklik ancak, genellikle önceden belirlenmiş bir tabak ya da çanak boyutu sınırlı sınırlı olabilir. Ayrıca luciferase ifadede mekansal varyasyon gösteren örnekleri Imaging bilgi kaybına yol açabilir bir Devresel ödeme kullanarak izlenen örnekleri kayma herhangi bir bilgi toplamak mümkün değildir. Ayrıca, devresel_ödeme ve ilişkili elektronik oksijen Standart hücre kültür kuluçka ortamına maruz kaldığında arıza eğilimli olduğundan, boyuna luciferase kayıt PMTs kullanarak her zaman sigara nemlendirilmelidir İnkübatörler içinde gerçekleştirilir. Sonuç olarak, hücre kültürü yemekleri gerekir kapalı hava-buharlaşma yoluyla nem kaybını önlemek için sıkı ve kültür medya bu nedenle 3 - ile tamponlanmış olmalıdır (N- morpholino) propanesulfonic asit (paspas) veya 4-(2-hydroxyethyl) -1- piperazineethanesulfonic asit (HEPES), in vivo fonksiyonları ve memeli doku kültüründe rutin olarak kullanılan sistem tampon CO2/bicarbonate yerine.
Bu sınırlamalar bir sonucu olarak PMTs tarafından bioluminescence ölçümü genellikle sıkı kısıtlamalar altında hücreler deneyler sırasında korunur koşulları üzerine yerleştirir. Bu sorunların üstesinden gelmek için ve aynı zamanda olası deneysel koşullar aralığı artırmak için biz bir su soğutulmuş elektron-çarparak eklenmesi tarafından uyarlanmış bir standart CO2/n2 170 L doku kültürü kuluçka makinesi kullanın Anti-sis optik ve dijital kontrol sıcaklık ve gaz düzeyleri ile şarj kuplajlı cihaz (EMCCD) kamera. Bu bir otomatik boyuna Luciferase gaz görüntüleme ve Temperature-Optimized kaydedici ya da timsah dönüşmüş bulunuyor. Kollarındaki görüntüleme, standart doku kültürü plakaların yüksek üretilen iş görüntüleme için her ikisi de önemli ölçüde artan esneklik için timsah sağlar (6 x 96 - veya 384-da kadar tabaklar aynı anda) ve standart dışı doku kültürü sistemleri, için de böyle derin hücreler olarak mikrosıvısal aygıt içinde büyüdü. Bu araç aynı zamanda oksijen koşullar altında ve değişken kontrolünü hem CO2 O2 kısmi basınç ve sıcaklık ile gerçekleşmesi için görüntüleme için sağlar.
İletişim kuralı aşağıdaki memeli hücre ve doku kültürü sistemleri (bundan böyle 'bioluminescence kuluçka' adlandırılır) bir timsah kullanarak kollarındaki kayıt için bir yöntemi açıklar. Bu, ancak, sistem kollarındaki görüntüleme ve aynı zamanda, bazı değişiklik, Floresan, diğer biyolojik sistemleri ve bağlamlarda bir dizi Imaging ile uygun olması gerekmektedir.

<table><tbody><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Hyclone FetalClone III Serum</td><td>GE Healthcare</td><td>SH30109.03</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher</td><td>21103049</td><td>basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A4919</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I1882</td><td></td></tr><tr><td>Superoxide Dismutase</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>Holo-transferrin</td><td>Calbiochem</td><td>616424</td><td></td></tr><tr><td>T3 (triiodo-L-thyronine</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C7518</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E9508</td><td></td></tr><tr><td>D (+)-Galactose</td><td>Sigma</td><td>G0625</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>Lipoic Acid</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Retinol Acetate</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Retinol, all trans</td><td>Sigma</td><td>95144</td><td></td></tr><tr><td>D,L-alpha-Tocopherol</td><td>Sigma</td><td>95240</td><td></td></tr><tr><td>D,L-alpha-Tocopherol acetate</td><td>Sigma</td><td>T3001</td><td></td></tr><tr><td>Sodium Bicarbonate Solution</td><td>Sigma</td><td>S8761-100ML</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Galaxy 170R incubator&amp;nbsp;</td><td>Eppendorf</td><td>CO17301001</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>D -(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>PHG0007</td><td></td></tr><tr><td>1 mm I.D. silicone tubing&amp;nbsp;</td><td>GE Healthcare</td><td>19-4692-01</td><td></td></tr><tr><td>Elbow luer connector</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Male luer fittings</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>Female luer fittings</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>&amp;micro;-slide luer I 0.6</td><td>Ibidi</td><td>80196</td><td></td></tr><tr><td>BD plastipak 20ml syringe</td><td>Becton Dickinson</td><td>300613</td><td></td></tr><tr><td>1mm I.D. ETFE tubing</td><td>GE Healthcare</td><td>18-1142-38</td><td></td></tr><tr><td>PF670462</td><td>Sigma</td><td>SML0795</td><td></td></tr><tr><td>B27 Supplement (50x)</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>iXon Ultra EMCCD camera</td><td>Andor</td><td>iXon 888</td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td>N/A</td><td></td></tr><tr><td>Prism 7.0</td><td>Graphpad Software</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>39DP</td><td></td></tr><tr><td>Heated neutral density filter&amp;nbsp;</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>Osmomat 030</td><td>Gonotech</td><td>Discontinued</td><td></td></tr><tr><td>300 mOsmol/kg calibration standard</td><td>Gonotech</td><td>30.9.0020</td><td></td></tr><tr><td>Measuring vessel</td><td>Gonotech</td><td>30.9.0010</td><td></td></tr><tr><td>Focusing cylinder</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>NE-1600 programmable syringe pump</td><td>Pump Systems inc.</td><td>NE-1600</td><td></td></tr><tr><td>Andor Solis Software</td><td>Andor</td><td>N/A</td><td></td></tr></tbody></table>

flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)

Luciferase tabanlı gazetecilere hücresel gen ifadesinin boyuna her ikisi için yaygın kullanılmayan ve biyolojik aktivitesini son nokta deneyleri. Sirkadiyen ritim araştırmada, örneğin, saat gen füzyon ile ateş böceği luciferase neden için sağlam ritimler birçok gün içinde kalıcı hücresel bioluminescence içinde olmaktadır. Teknik sınırlamalar photomultiplier tüp (PMT) ile ilişkili veya geleneksel mikroskobu tabanlı yöntemler bioluminescence miktar için genellikle hücre ve dokuların sırasında oldukça fizyolojik koşullarda muhafaza edilmesi talep var. kayıt, duyarlılık ve işlem hacmi arasında bir denge ile. Burada, uzun vadeli bioluminescence görüntüleme yüksek hassasiyet ve hangi kültür koşullar, değişken gaz ve nem kontrolü de dahil olmak üzere geniş bir dizi çekmek ve bu birçok kabul eden işlem hacmi ile farklı sağlar bir arıtma önceki yöntemlerden raporu doku kültürü tabak ve yemekler. Bu otomatik boyuna luciferase gaz ve sıcaklık optimize Kaydedici (timsah) ayrıca Imaging mekansal varyasyon gözlem luciferase ifadede hücre monolayer veya kolayca geleneksel tarafından gözlenen cant doku üzerinden sağlar yöntemleri. Biz nasıl timsah varolan yöntemleri ile karşılaştırıldığında luciferase aktivite tespiti için büyük ölçüde artan esneklik sağlar vurgulayın.

Bu makalede bir timsah (bioluminescence kuluçka) kullanarak memeli hücrelerinin kollarındaki görüntüleme için bir protokol özetlenmektedir. Bu teknik görüntüleme kollarındaki sistemleri zaman için fiziksel kurulum ve ekstraselüler koşullara esneklik sağlar. Basit statik doku kültürü (şekil 1, ek Video 1) ve derin hücre kültürü (Şekil 2, ek Video 2) yöntemleri açıklanan, ama birçok diğer kurulumları bu sistemi kullanarak yansıma. Tüm veri Bölüm 7'de açıklanan yöntemleri kullanarak sayısal.
Şekil 1A PER2::LUC fibroblastlar4içeren 6 x 96-şey plakalar kayıt bioluminescence video gösterilmektedir. Bunlar bu deneme için gerekli değildi olarak en dıştaki wells hücreleri içermez. Hücreleri farklı sıcaklık sürüklenme, mademki 12 h 37 ° C 72 h veya converse (37 ° c 32 ° c 72 h 12 h ardından 12 h), ardından 32 ° C'de 12 h her iki sıcaklık döngüsünü geçmesi kayıt için sürekli 37 ° C'de tutulan önce geçmiştir. 60 dk Etkilenmeler her saat çekildi. Bu koşullar iki şekil 1Badımında sayısal.
Şekil 2A derin doku kültürü için bir sistemi kurulumunun şematik gösterir. Bu boru ile bağlı iki kanal slayt oluşur. Medya hücreler arasında bir enjektör pompası tarafından tahrik edilmektedir. Bu slaytları ilk gaz geçirgen kabarcık tuzak (arabellek slayt) olarak işlem görür ve ikinci ile hiçbir hücre içerir bioluminescence kaydedilir hücreleri içeren. Bu sistem video kaydı bir temsilcisi şekil 2Biçinde gösterilir. 15 dk Etkilenmeler her 15 dakikada çekildi. Burada, hücreleri standart perfüzyon koşullar altında veya kazein kinaz inhibitörü daha önce sirkadiyen süresini uzatmak ve kültürlü saat gen ifade ritimler genliğini azaltmak için gösterilmiştir PF670462 huzurunda korunur memeli hücre14. PER2::LUC ifade etkisi şekil 2C içinde (üst panel) uyuşturucu şekil 2C içinde (alt paneli), gösterilen standart statik hücre kültür koşullar altında aynı konsantrasyonu ile dönemin miktar ile tedavi hücrelere karşı gösterilen şekil 2Bolarak gösterilir. Bu net PF670462 ile tedavi PER2::LUC ifadesi altında her iki koşul kümesinden birini etkiler bu. Hücreleri periosteum koşulları Haritayı dönem yaklaşık 3 h (3 ±0.9 h) uzatma altında PF670462 ile tedavi ederken, ancak, hücreleri statik koşullarda ilaç ile tedavi daha fazla dönem için bir süre uzatma gösterir &#62; 48 saat. Bu damped kosinüs tarafından Bölüm 7'de açıklandığı gibi uygun olabilir (ekstra toplamı, kareler F test karşı düz bir çizgi, p &#60; 0.0001). İlginçtir, dönem perfüzyon altında uzatma büyüklüğü Bu vivo içinde14gözlenen çok daha yakın.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg"> 
Şekil 1: veri örneği. (A)örnek bioluminescence ölümsüzleştirdi PER2::LUC 6 x 96 dan video görüntüsünü de bioluminescence kuluçka makinesine tabaklar. Kuyu sayısal için tamamlayıcı Video 1sarı vurgulu olarak gösterilmiştir. (B) miktar bioluminescence A. üzerinden İki setleri 3 hücre entrained Anti-PER2 protein ifade aşamalarını üretmek için birbirleriyle phasic sıcaklık döngüsü (12 saat 32 ° C'de; 12 saat 37 ° C'de) kullanarak (n = 6 ± SEM demek). <a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig1v2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56623/56623fig2v2.jpg"> 
Resim 2: CK1δ inhibitörleri ile perfüzyon. (A)şeması perfüzyon sistemi. (B) örnek PER2::LUC kayıt bioluminescence anlık görüntü altında perfüzyon hücreleri. Miktar bir örnek alan Sarı renkle vurgulanır. PER2::LUC perfüzyon altında ve statik koşullarda ve 3 µM CK1 inhibitörü PF670462 olmadan fibroblast biyolüminesans (C) miktar (n = 3, ± SEM demek). Bioluminescence için minimum ve maksimum değerleri normalleştirilmiş. (D) analiz dönemi (Two-way ANOVA, Holm-Sidak'ın birden fazla karşılaştırma testi).<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig2v2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bileşen</td>
			<td>Son orta konsantrasyonu (mikron)</td>
			<td>Hisse senedi (mg/mL)</td>
			<td>400 mL NS21 için</td>
		</tr>
		<tr>
			<td>Sığır albümin</td>
			<td>37</td>
			<td>-</td>
			<td>50 gr</td>
		</tr>
		<tr>
			<td>Katalaz</td>
			<td>0,01</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Glutatyon</td>
			<td>3.2</td>
			<td>-</td>
			<td>20 mg</td>
		</tr>
		<tr>
			<td>İnsülin</td>
			<td>0,6</td>
			<td>10</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Süperoksit Dismutaz</td>
			<td>0.077</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>0.062</td>
			<td>-</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>T3 (triiodo-L-thyronine)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20 ΜL</td>
		</tr>
		<tr>
			<td>L-karnitin</td>
			<td>12</td>
			<td>-</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>Etanolamin</td>
			<td>16</td>
			<td>Sıvı (1 g/ml)</td>
			<td>20 ΜL</td>
		</tr>
		<tr>
			<td>D (+)-galaktoz</td>
			<td>83</td>
			<td>-</td>
			<td>300 mg</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>183</td>
			<td>-</td>
			<td>322 mg</td>
		</tr>
		<tr>
			<td>Sodyum selenit</td>
			<td>0.083</td>
			<td>1</td>
			<td>280 ΜL</td>
		</tr>
		<tr>
			<td>Corticosterone</td>
			<td>0.058</td>
			<td>2</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Linoleik asit</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Linolenik asit</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Lipoik asit</td>
			<td>0,2</td>
			<td>4.7</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Progesteron</td>
			<td>0,02</td>
			<td>3.2</td>
			<td>0,04 mL</td>
		</tr>
		<tr>
			<td>Retinol asetat</td>
			<td>0,2</td>
			<td>20</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Retinol, tüm trans</td>
			<td>0,3</td>
			<td>10</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>D, L-alfa-tokoferol</td>
			<td>2.3</td>
			<td>100</td>
			<td>0. 2 mL</td>
		</tr>
		<tr>
			<td>D, L-alfa-tokoferol asetat</td>
			<td>2.1</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
	</tbody>
</table>
Tablo 1: NS21 hazırlık.
Ek Video 1: örnek video bioluminescence iyi plakalar üzerinden. Örnek video anlık görüntüsünü bioluminescence 6 x 96 iyi plakaları bioluminescence kuluçka ölümsüzleştirdi PER2::LUC üzerinden. Sayısal için kuyu Sarı renkle vurgulanır. <a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_1A_Revision-1.avi">Bu dosyayı indirmek için buraya tıklayınız.</a>
Ek Video 2: PER2::LUC kayıt bioluminescence örnek video görüntüsünü hücreleri perfüzyon altında. Miktar bir örnek alan Sarı renkle vurgulanır. <a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_2B_Revision-1.avi">Bu dosyayı indirmek için buraya tıklayınız.</a>

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetik olarak kodlanmış luciferase gen ekspresyonu popüler bir non-invaziv muhabir var. Gaz ve sıcaklık optimize Kaydedici (timsah) Imaging otomatik bir boyuna luciferase çok çeşitli koşullar altında kollarındaki hücrelerden Boylamsal kayıt sağlar. İşte nasıl timsah sirkadiyen ritim araştırma bağlamında kullanılabilir göstermektedir.

Kollarındaki gazetecilere bir otomatik boyuna Luciferase gaz ve sıcaklık optimize Kaydedici (timsah) Imaging kullanarak esnek ölçüm

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

flexible-measurement-bioluminescent-reporters-using-an-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

7.8K Görüntüleme.  MRC Laboratory of Molecular Biology. Genetik olarak kodlanmış luciferase gen ekspresyonu popüler bir non-invaziv muhabir var. Gaz ve sıcaklık optimize Kaydedici (timsah) Imaging otomatik bir boyuna luciferase çok çeşitli koşullar altında kollarındaki hücrelerden Boylamsal kayıt sağlar. İşte nasıl timsah sirkadiyen ritim araştırma bağlamında kullanılabilir göstermektedir.

Video: Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Farelerde Görüntü Bakteriyel Enfeksiyonlar Lusiferaz Kullanımı

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

İmmünoloji ve Enfeksiyon

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Vivo Çift Yüzey Bioluminescent Imaging&#39;de

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Tıp

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

Situ Ölçüm ve korelasyon hücre yoğunluğu ve kollarındaki bakterilerin ışık emisyon

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Biyokimya

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Membran Biyogenez bir Lusiferaz Tabanlı Reporter Gen Testi ile incelenmesi

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biyoloji

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Murin Nörogenezi Sırasında Çentik Sinyal Dinamiğinin Gerçek Zamanlı Biyolüminesans Görüntülemesi

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Gelişim Biyolojisi

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Lusiferaz Biyoparlaklık Gazeteciler kullanma Gen Ekspresyonunun Hücre-özerk Sirkadiyen Saat Ritimleri İzlenmesi

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Sitotoksisite Sürekli ve gerçek zamanlı izlenmesi için özerk Bioluminescent Memeli Hücre

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Sirkadiyen Ritim Çalışmaları için İnsan Bağırsak Enteroidlerinin Optimize Edilmesi

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Yazar Spotlight: Çok Hücreli Sistemlerde Sirkadiyen Ritimlerin İn Vitro Araştırmaları

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Organoidlerde Biyolüminesans Raportörü ile Sirkadiyen Salınımların İzlenmesi

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Kollarındaki gazetecilere bir otomatik boyuna Luciferase gaz ve sıcaklık optimize Kaydedici (timsah) Imaging kullanarak esnek ölçüm

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