Name: detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system
Uploaded: 2018-04-09T12:30:00
Description: Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Chi-Chung Chen üzerinden Bakanlığı tıbbi araştırma, Chi Mei Tıp Merkezi (Tainan, Tayvan) fare yardım için enfeksiyon ve ulusal Cheng Kung Üniversitesi laboratuvar hayvan merkezinden destek anıyoruz. Bu eser bilim Bakanı tarafından desteklenir ve Teknoloji (en) CC için (en 104-2321-B-006-019, 105-2321-B-006-011, and106-2321-B-006-005) verir.

Dikkat: EHEC O157: H7 Biyogüvenlik seviye 2 (BSL-2) olan patojen merkezleri hastalık kontrol ve Önleme (CDC) Biyogüvenlik talimat (https://www.cdc.gov/) göre. Bu nedenle, tüm deneysel prosedürler EHEC içeren BSL-2 tesisinde gerçekleştirilmelidir. Laboratuvar mont ve eldiven giymek deney yaparken. Bir Sertifikalı Biyogüvenlik içinde dolap (BSC) çalışır. Deneysel kürsüye önce ve deneysel bir işlem ile % 70 etanol sonra dezenfekte. Bütün aletleri veya ekipman kişi (veya potansiyel kişi) EHEC % 70 et...

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Bu luciferase plazmid ile dönüştürülmüş EHEC onun Yerelleştirme Ana bilgisayarlar veya gen ifade vivo içinde8,11,12incelemek için kullanılan bildirilmiştir. Aşağıda gösterildiği fare modeli ayrıca fare ana bilgisayar8' bulmak belgili tanımlık kolonize EHEC zamanlama ve yerelleştirme bildirilmiştir. Yine de, biz, farelere EHEC aşılama intragastrically yönetmek ve kolların...

EHEC O157: H7 nedenleri ishal1, HS2, HUS3ve hatta Akut böbrek yetmezliği4 kontamine su veya gıda ile bir patojen var. EHEC patojenik bir enterobacterium olduğunu ve insanların1gastrointestinal sistem colonizes. EHEC ilk uygun zaman ana bağırsak epiteli, kolonizasyon faktörler bir bağlama inducing ve daha sonra zorlamak için (A/E) lezyon effacing moleküler bir şırınga işlev gören tür III salgı sistemi (T3SS) ile ana hücreleri içine enjekte yapışma (kolonizasyon)5. Bu genlerin A/E lezyon oluşumunda yer alan Enterocytes efasman (LEE) patojenitesi ada5locus tarafından kodlanır.
Bioluminescence olduğunu ışık üreten kimyasal reaksiyon, görünür ışık6oluşturmak için onun substrat biyoluminesans hangi luciferase tromboksan. Enzimatik bu işlem genellikle oksijen veya adenozin trifosfat (ATP)6varlığını gerektirir. (BLI) görüntüleme bioluminescence araştırmacılar canlı hayvan7görselleştirme ve ana bilgisayar-patojen etkileşimlerin niceleme sağlar. Saçınıza canlı hayvan bakteriyel enfeksiyon döngüsünde onlar göç ve farklı dokular7istila kollarındaki bakteri izleyerek karakterize olabilir; Bu enfeksiyon dinamik bir ilerleme ortaya koymaktadır. Ayrıca, hayvanlarda bakteri yükü kollarındaki sinyal8' e ilişkilidir; Böylece, deneysel hayvan patolojik şartları basit ve doğrudan bir şekilde tahmin etmek için uygun bir göstergedir.
Burada kullanılan plazmid luciferase operon, bakteri, kendi luciferase substrat7,9kodlar Photorhabdus luminescens olan luxCDABE, yer. Bu luciferase ifade plazmid bakteri dönüştürerek, canlı hayvan kollarındaki bu bakteri gözlemleyerek kolonizasyon ve enfeksiyon işlemleri izlenebilir. Genel olarak, alma ve bioluminescence etiketli bakteri araştırmacılar bakteri numaraları ve konumu, bakteriyel canlılığı antibiyotikler/tedavi ve bakteri gen ekspresyonu enfeksiyon/kolonizasyon6, ile izlemek izin 7. çok sayıda patojen bakteri enfeksiyonu onların enfeksiyon döngüsü ve/veya gen ifade incelemek için luxCDABE operon express bildirilmiştir. Bu bakterilerin uropathogenic E. coli10, EHEC8,11,12,13, enteropathogenic de dahil olmak üzere, E. coli (EPEC)8, Sitrobacter rodentium14,15, Salmonella typhimurium16, Listeria Monositogenez17, Yersinia enterocolitica18,19, ve Vibrio cholerae20, dokümante edilmiştir.
EHEC kolonizasyon vitro ve in vivo21,22,23çalışma kolaylaştırmak için çeşitli modeller geliştirilmiştir. Ancak, EHEC kolonizasyon vivo içindeçalışmaya uygun hayvan modellerin eksikliği ve böylece ayrıntılar ortaya çıkan bir yetersizliği olduğunu. EHEC kolonizasyon mekanizması vivo içindeçalışma kolaylaştırmak için gözlemlemek ve non-invaziv bir yöntem canlı hayvan EHEC kolonizasyon ölçmek için hayvan modelleri oluşturmak için değerlidir.
Bu el yazması kollarındaki ifade sisteminin EHEC kolonizasyon zaman içinde yaşayan ana bilgisayarları izlemek için kullandığı bir fare-EHEC kolonizasyon modeli açıklar. Fareler intragastrically bioluminescence etiketli EHEC ile aşılanmış ve kollarındaki sinyal farelerde sistem13Imaging bir non-invaziv vivo içinde ile algılanır. Sonra 2 gün sonra 2 gün enfeksiyon sonrası bu bakteri konak bağırsaklarda kolonize önerilen enfeksiyon sonrası önemli kollarındaki sinyalleri onların bağırsaklarda gösterdi EHEC bioluminescence etiketli fareler bulaşmış. Ex vivo görüntü verilerini bu kolonizasyon özellikle çekum ve kolon farelerin olduğunu gösterdi. Bu fare-EHEC modelini kullanarak, kollarındaki EHEC kolonizasyon yaşam ana bilgisayarında bir görüntüleme sistemi daha fazla anlayış teşvik edebilir enterik bakteri kolonizasyonu detaylı mekanizmaları incelemek için in vivo tarafından tespit edilebilir EHEC kaynaklı fizyolojik ve patolojik değişiklikler.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Shaker incubator</td>
			<td style="width:175px;">YIH DER</td>
			<td style="width:143px;">LM-570R</td>
			<td style="width:167px;">bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Orbital shaking incubator</td>
			<td style="width:175px;">FIRSTEK</td>
			<td style="width:143px;">S300</td>
			<td>bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">pBSL180</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of nptII gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pAKlux2</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of luxCDABE operon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T&#38;A Cloning Kit</td>
			<td>Yeastern Biotech</td>
			<td style="width:143px;">FYC001-20P</td>
			<td>use for TA cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nsi I</td>
			<td style="width:175px;">NEB</td>
			<td>R0127S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sca I</td>
			<td style="width:175px;">NEB</td>
			<td>R0122S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I-HF</td>
			<td style="width:175px;">NEB</td>
			<td>R0133S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sma I&#160;</td>
			<td style="width:175px;">NEB</td>
			<td>R0141S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:175px;">NEB</td>
			<td style="width:143px;">M0202S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ex Taq</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">10X Ex Taq Buffer</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP Mixture&#160;</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">PCR machine</td>
			<td style="width:175px;">applied Biosystem&#160;</td>
			<td style="width:143px;">2720 thermal cycler&#160;</td>
			<td>&#160;for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:175px;">SIGMA</td>
			<td style="width:143px;">G5516-1L</td>
			<td>use for bacteria stocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NaCl</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">31434-5KG-R</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tryptone</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1612.00</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Yeast Extract powder</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:143px;">23547-1 KG</td>
			<td>chemical for making LB medium, 5 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Agar</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1802.00</td>
			<td>chemical for making LB agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">kanamycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">K4000-5G</td>
			<td>antibiotics, use for seleciton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">streptomycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">S6501-100G</td>
			<td>antibiotics, eliminate the microbiota in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">EDL933 competent cell</td>
			<td style="width:175px;">Homemade</td>
			<td style="width:143px;"></td>
			<td>method is on supplemental document&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporator</td>
			<td style="width:175px;">MicroPulser</td>
			<td style="width:143px;"></td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporation Cuvettes</td>
			<td style="width:175px;">Gene Pulser/MicroPulser</td>
			<td style="width:143px;">1652086</td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">High-speed centrifuge</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">Avanti, J-26S XP</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JA25.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JLA10.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td>Beckman Coulter</td>
			<td>REF357003</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td style="width:175px;">Thermo Fisher scientific</td>
			<td style="width:143px;">3141-0500</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">eppendorf biophotometer plus&#160;</td>
			<td>eppendorf</td>
			<td>AG 22331 hamburg</td>
			<td>for measuring the OD600 value of bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">C57BL/6 mice&#160;</td>
			<td colspan="2">Laboratory Animal Center of NCKU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">lab coat, gloves</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>for personnel protection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane&#160;</td>
			<td>Panion &#38; BF Biotech Inc.</td>
			<td style="width:143px;">G-8669</td>
			<td>for mice anesthesia, pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Reusable 22 G ball-tipped feeding needle</td>
			<td style="width:143px;">&#966;0.9 mm X L 50 mm</td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical&#160; scissors&#160;</td>
			<td></td>
			<td style="width:143px;"></td>
			<td>use for mice experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xenogen IVIS 200 imaging system</td>
			<td>Perkin Elmer</td>
			<td>IVIS spectrum</td>
			<td>use for bioluminescent image capture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Living Image Software</td>
			<td>Perkin Elmer</td>
			<td style="width:143px;">version 4.1</td>
			<td>use for quantifying the image data</td>
		</tr>
	</tbody>
</table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

Enterohemorrhagic E. coli (EHEC) O157: H7, hangi o causesdiarrhea, Hemorajik kolit (HS), gıda kaynaklı patojen olduğunu ve hemolitik üremik Sendromu (HUS), kolonize insanlar bağırsak için. EHEC kolonizasyon içinde vivo detaylı mekanizması çalışmaya EHEC kolonizasyon ölçmek ve izlemek için hayvan modelleri olması esastır. Burada bir fare-EHEC kolonizasyon modeli kollarındaki ifade plazmid EHEC izlemek ve yaşam ana EHEC kolonizasyon ölçmek için dönüşüm tarafından göstermektedir. Bioluminescence etiketli EHEC ile aşılanmış hayvanlar yoğun kollarındaki sinyalleri farelerde algılama tarafından görüntüleme sistemi bir non-invaziv vivo içinde ile göstermek. Sonra 1-2 gün enfeksiyon sonrası, kollarındaki sinyalleri hala enfekte hayvanlarda da EHEC konaklarda en az 2 gün için kolonize gösterir tespit edilemedi. Biz de bu kollarındaki EHEC bulun ki fare Bağırsaklarda, özellikle çekum ve iki nokta üst üste, için ex vivo imge--dan göstermek. Bu fare-EHEC kolonizasyon model EHEC kolonizasyon mekanizma mevcut bilgi ilerlemek için bir araç olarak hizmet verebilir.

Bioluminescence etiketli EHEC yönetilen (~ 109 bakteri hücreleri) 6 - hafta eski kadın C57BL/6 farelere oral gavaj tarafından. EHEC farelere 1s içinde sözlü aşı sonra hayvanlar kollarındaki sinyali VIVO içinde görüntüleme sistemi Şekil 7gösterildiği gibi tarafından incelenmiş. Sonuçlar sonda ile besleme farelerde bioluminescence etiketli EHEC ile güçlü bir kollarındaki sinyal gösterdi. Biz 2 gün sonrası enfeksiyo...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Bir fare modeli enterohemorrhagic bioluminescence etiketli bakteri kullanarak E. coli (EHEC) kolonizasyon için detaylı bir protokol sunulmuştur. Kollarındaki bu bakteriler tarafından bir non-invaziv görüntüleme sistemi içinde canlı hayvan vivo içinde tespiti geçerli anlayışımız EHEC kolonizasyon ilerletebilirsiniz.

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Research

JoVE Journal

Immunology and Infection

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Lökosit Homing Göç ve non-invaziv Görüntüleme In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Zebra balığı Larva yılında Dissemine Kandidiyazis non-invaziv Görüntüleme

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Neuropathogenic olmayan İnvaziv Modeli<em&gt; Escherichia coli</em&gt; Yenidoğan Sıçan Enfeksiyon

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Tek adımlı olumsuz Kromatografik arıtma<em&gt; Helicobacter pylori</em&gt; Nötrofil-aktive protein aşın<em&gt; Escherichia coli</em&gt; Toplu Modunda

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

B Grubu bir murin modelinde<em&gt; Streptococcus</em&gt; Vajinal Kolonizasyon

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invaziv<emIn vivo</em&gt; Aktive Edilebilir Floresan Görüntüleme Ajanını Kullanan İnflamatuvar MMP Aktivitesinin Flüoresan Optik Görüntülemesi

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Farelerde Gardnerella vaginalis Mesane Maruziyeti Tarafından Tetiklenen Tekrarlayan Escherichia coli İdrar Yolu Enfeksiyonu

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Meningitik Escherichia Coli ile Enfekte Olan Nötrofillerde Reaktif Oksijen Türlerinin Gerçek Zamanlı Nicelemesi

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...