Name: biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans
Uploaded: 2015-03-19T16:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Bu çalışma, Ulusal Bilim Vakfı (Ödül # 0.920.274) ve Güney Carolina Deney İstasyonu Projesi SC-1.700.340 dan ödülle tarafından desteklenmiştir. Clemson Üniversitesi Deney Station Bu kağıt isTechnical Katkı sayılı 6283. Yazarlar bu son protokol ve yazının eleştirel okuma Dr Cheryl Ingram-Smith, Katie Glenn, Grace Kisirkoi gelişiminde onun yararlı tavsiyeler Dr. Lukasz Kozubowski teşekkür ederim.

NOT: Bu protokolün genel şeması Şekil 1&#39;de özetlenmiştir. 1. C. neoformans Hazırlık <ol><li> Her bir dönüşüm reaksiyonu için, C 2-3 mi O / N kültürünün yetiştirilmesi 30 ° C&#39;de YPD besiyeri içinde neoformans 250 rpm &#39;de çalkalanarak. </li><li> 10 ° C&#39;de 900 x g&#39;de 5 dakika boyunca O / N kültürü santrifüj ve süpernatant atılır. </li><li> Maya Peptonlu Dekstroz (YPD) orta 300 ul her hücre pelletini. </li><li> Cam boncuklar kullanılarak, yavaşça 1 M sorbitol içeren YPD agar üzerine yıkandı hücre süspansiyonu 300 ul yayıldı. </li><li> Plakalar 3-4 saat için çevre sıcaklığında kurumaya bırakın. </li></ol> 2. altın mikro taşıyıcı preparatı <ol><li> GKD 2 O, 1 ml 0.6 um altın parçacıkları, 0.25 g yeniden süspanse 900 xg&#39;de 1 dakika boyunca santrifüj boncuk pelet ve süpernatan. </li><li> % 100 etanol içinde 1 ml altın boncuk tekrar. &lt;/ Li&gt; <li> 4 tüpler, her ul 250 içine boncuk dağıtın ve% 100 etanol 750 ul ekleyin. </li><li> 4 ° C&#39;de saklayın altın boncuk tümbölenleri. </li></ol> 3. DNA hazırlanması <ol><li> % 100 etanol kullanılarak forseps onları daldırarak turuncu macrocarrier biolistik diskleri hazırlayın. Kuruması için Drierite içeren geniş bir petri içine yerleştirin diskler (DRIERIT diskleri temas etmediğinden emin olun). </li><li> Kuruduktan sonra, (daha önce% 100 etanol ile sildi) gümüş disk sahipleri içine macrocarrier diskleri basın. </li><li> Dönüşüm başına yaklaşık 1.5 ml mikrosantrifüj tüpü, bir tüp içine (aşama 2&#39;de tarif edildiği gibi hazırlanır) Burulma altın boncuklar ve kısım 12 ul. </li><li> Için her bir tüpe ekleyin: 2 DNA&#39;sının ug (tercihen 1 ug DNA / ml 2 ul), 10 ul 2.5 M CaCI2 ve 2 ul 1 M spermidin serbest baz. </li><li> Adım 3.4 olarak ama hiçbir DNA ile bir negatif kontrol kurmak. </li><li> Vorteks her boru ve çevre sıcaklığında inkübe5 dak. Yavaşça bu inkübasyon sırasında yerleşmiş boncuk tekrar süspansiyon bazen her tüp fiske. </li><li> 30 saniye için 225 x g&#39;de santrifüj tüpleri DNA-kaplı altın boncukların topaklanması için. Dikkatle (pipetle veya aspirasyon ile) süpernatant kaldırmak ve atın. </li><li> Yavaş yavaş yukarı ve aşağı pipetleme tamamen% 100 etanol 600 ul süspanse boncuk. </li><li> Ambalaj olmadan boncuk pelet 30 saniye için 225 xg&#39;de tüpler Spin. Dikkatle çıkarın ve süpernatant atın. </li><li> Yavaş bir şekilde pipetli yukan aşağı% 100 etanol içinde 8 ul DNA ile kaplanmış altın boncukları yeniden süspanse edin. </li><li> 1 cm çapında biolistik diskin merkezi üzerine DNA kaplı altın boncuk Pipet ve kurumasını bekleyin. Not: Biyolistik diskin merkezinden görünür bir kuru, altın daire altın parçacıkları yeterli bir konsantrasyon olduğunu gösterir. Not: DNA-kaplı altın parçacıkları ile yüklenen makro-taşıyıcı disklerin hemen gen tabancası ile kullanım için hazırdır. </li></ol> 4. ÇGen Tabancası perating <ol><li> Vakum pompası açın. </li><li> Yaklaşık 2.200 psi basınç kadar saat yönünün düğmeyi çevirerek helyum gazı açın manometre ulaşılır. </li><li> Soldaki kırmızı anahtarı çevirerek gen tabancası açın. </li><li> Vakum 15 saniye içinde 28 inç Hg ulaşacak, böylece vakum ve havalandırma için debileri ayarlanmış olduğundan emin olun. </li><li> Kopma diski ve makrotaşıyıcı arasındaki mesafe yaklaşık 3/8 inç olduğundan emin olun. </li><li> Etanol ile silerek tüm odasını temizleyin. </li><li> % 100 etanol içinde kopma diskleri Batmak. Steril bir yüzey üzerinde kurumaya bırakın (örneğin, Petri kabı). </li><li> Rüptür disk yuvasını gevşetmek için bir tork anahtarı kullanın. Tutucu içine temiz bir kopma diski takın. Geri yerine kopma disk tutucu vida ve sağa kez çevirerek tork anahtarı ile sıkın. NOT: Kesilme diskleri her sürgün aşağıdaki değiştirilecektir. </li><li> Th Batmak% 100 etanol içinde E örgü ekranları. Steril bir yüzey üzerinde kurumaya bırakın (örneğin, Petri kabı). </li><li> Kuruduktan sonra, beyaz plastik montaj plakası üzerinde bir yıkanmış elek yerleştirin. Disk odasına aşağı macrocarrier disk tutucu DNA tarafı yerleştirin. Gümüş kapağı vidalayın ve yüksek yuvasına montaj plakasını yerleştirin. NOT: örgü ekranı her çekimlerden sonra değiştirilecektir. </li><li> Alt plaka üzerinde 1 M sorbitol içeren bir YPD agar plaka koyun. </li><li> Kamara kapıyı kapattı ve yerine kilitleyin. </li><li> Itin ve vakum meşgul ve vakum 28 inç Hg ulaşmak için izin orta kırmızı anahtarını tutun. Uygun vakum seviyesi ulaşıldığında, aşağı pozisyona getiriniz. Hazır olduğunuzda, yangın sağdaki kırmızı düğmeyi basılı tutun. Rüptür disk attığında, hemen yangın düğmesini bırakın ve 0 psi odasına havalandırma için orta pozisyona orta kırmızı anahtarı itin. </li><li> Rüptür disk enkaz temizleyin ve gen silahı kapatın. Sonra heliu kapatmakdüğmeyi saat yönünde çevirerek, ve nihayet, vakum pompası kapatmak tarafından m gazı. </li></ol> 5. Kaplama Dönüştürülmüş hücreler <ol><li> Dönüşüm plakalar hücre kurtarmak için izin vermek için, 4 saat boyunca oda sıcaklığında bekletin. </li><li> Plaka üzerine YPD Pipet 700 ul. Yavaşça bir steril 1.5 ml mikrofuge&#39;de tüp içine agar ve pipet sıvı kapalı hücreleri kazımak için bir hücre kazıyıcı kullanın. Tüm hücreleri plaka elde edilmiştir sağlamak için bu adımı tekrarlayın. </li><li> 30 saniye için 225 x g&#39;de topak hücrelere. Süpernatantı çıkarın ve atın. </li><li> YPD 500 ul pelletini. </li><li> Pipet 100 YPD + antibiyotik plakalar merkezi üzerine hücre süspansiyonu ul ve cam boncuklar kullanarak yayılır. </li><li> 3-4 gün boyunca oda sıcaklığında ters plakaları bırakın. Koloniler görünür gibi, yeni YDP + antibiyotik plakalar üzerine yama. </li></ol> PCR için 6. genomik DNA izolasyonu NOT: Bu reaktif kullanılarak modifiye edilmiş bir versiyonudurDNA saflaştırma kiti s (Malzeme Tablo bakınız). <ol><li> C her biri 5 ml&#39;lik bir kültür büyütün 30 ° C&#39;de YPD sıvı içinde neoformans transformantlar 250 rpm&#39;de göre O / N çalkalanarak. </li><li> 900 x g&#39;de hücrelerin 3 ml Pelet, ve çekirdekler lizis çözeltisinin 600 ul süspansiyon haline getirin. </li><li> 0,5 mm asit 200 ul yeni bir 1.5 mL mikrosantrifüj tüpüne süspansiyonu ekleyin cam boncuk yıkanmıştır. </li><li> Buz üzerinde ortam sıcaklığında, serin tüp mini beadbeater 45 sn için homojenize ve tekrarlayın. </li><li> Örnek 2 dakika süreyle buz üzerine yerleşebilecek ve yeni bir 1.5 ml&#39;lik tübe süpernatan aktarmak için izin verir. 20 saniye boyunca kuvvetli bir şekilde ve vorteks (süpernatan geri kazanılmış her 600 ul için 100 ul), her bir tüpe, protein çökeltme çözeltisinin 200 ul ekle. </li><li> Numuneler, 5 dakika için buz üzerine yerleşmek ve 3 dakika süre ile 11,000 x g&#39;de santrifüj izin verin. </li><li> RT izopropanol 300 ul içeren bir temiz 1,5 ml tüp süpernatant aktarın. Yavaşça tersine çevirerek karıştırın. </li><li>Santrifüj örnekleri 2 dakika boyunca 11.000 xg&#39;de, dikkatle süpernatant kaldırmak ve kağıt havlu üzerine tüpleri boşaltın. </li><li> Her bir tüp, RT% 70 etanol içinde 300 ul ekleyin ve yavaşça pelet yıkama için ters. </li><li> Dikkatle 11,000 2 dakika xg, ve Santrifüj örnekleri etanol kaldırmak. </li><li> Temiz bir kağıt havlu üzerine tüp boşaltın ve 10-15 dakika boyunca kuru hava pelet izin. </li><li> DNA rehidrasyon solüsyonu 50 ul, her topak ve vorteks RNaz çözeltisi 1.5 ul ekleyin. </li><li> 5 saniye boyunca santrifüj örnekleri kapağından sıvı kaldırmak için. </li><li> 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> 1 saat boyunca 65 ° C&#39;de örneklerin inkübe etmek suretiyle DNA rehidrate. </li><li> 260 nm&#39;de absorbans spektrofotometrik olarak ölçülmesiyle DNA niceliğini (1.0 A 260 okuma ~ 50 ug / ml, çift dallı DNA eşdeğerdir) ve her PCR reaksiyonunda 200 ng kadar kullanılabilir. </li></ol> 7. RNA İzolasyon içinTranscriptase-PCR Ters. <ol><li> Bir RNA arıtma kiti (Malzeme Tablo bakınız) kullanarak, bir minibeadbeater kullanarak maya hücrelerinden RNA izole etmek için üreticinin talimatlarını izleyin. </li><li> 260 nm&#39;de absorbans ölçülerek RNA konsantrasyonunu belirlemek (1.0 A 260 okuma eşdeğerdir mi ~ 40 ug / tek sarmallı RNA). </li><li> RT-PCR kiti (Malzeme Tablo bakınız) kullanarak, yaklaşık 1 ug RNA RT-PCR reaksiyonları kurmak için üreticinin talimatlarını izleyin. Bu çalışmada elde edilen sonuçlar için Tablo 1 &#39;de listelenen primerleri kullanılır. </li></ol>

<ol>
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We have nothing to disclose.

Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 &#181;g. However, the volume of DNA added to the gold beads can be increased in the chance the...

Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide 1. Acetate has been shown to be a major fermentation product during cryptococcal infection 2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection 5. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including&#160;C. neoformans&#160;6.
To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation 7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus 9, it is not useful in creating stable homologous transformants 8. Only&#160;biolistic&#160;delivery using a gene gun&#160;provides&#160;an effective means to transform&#160;linear&#160;DNAs&#160;that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable 9. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant 10. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered 7,8,11. 
This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the&#160;ack&#160;locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.

<table><tbody><tr><td>0.6 &amp;mu;m gold beads</td><td>Bio-Rad</td><td>165-2262</td><td>http://www.bio-rad.com</td></tr><tr><td>Spermadine-free base</td><td>Sigma- Aldrich</td><td>S0266</td><td>https://www.sigmaaldrich.com</td></tr><tr><td>G418 - Sulfate (Neomycin)</td><td>Gold Biotechnology</td><td>G-418-10</td><td>www.goldbio.com</td></tr><tr><td>Hygromycin</td><td>Gold Biotechnology</td><td>H-270-1</td><td>www.goldbio.com</td></tr><tr><td>1350 psi Rupture Discs</td><td>Bio-Rad</td><td>165-2330</td><td>http://www.bio-rad.com</td></tr><tr><td>Stopping Screens</td><td>Bio-Rad</td><td>165-2336</td><td>http://www.bio-rad.com</td></tr><tr><td>Macrocarriers discs</td><td>Bio-Rad</td><td>165-2335</td><td>http://www.bio-rad.com</td></tr><tr><td>YPD Broth</td><td>Becton Dickinson &amp;amp; Co.</td><td>242820</td><td>www.bd.com</td></tr><tr><td>Agar</td><td>Becton Dickinson &amp;amp; Co.</td><td>214530</td><td>www.bd.com</td></tr><tr><td>Sorbitol</td><td>Fisher Scientific</td><td>BP439</td><td>http://www.fishersci.com</td></tr><tr><td>PDS-1000/He System</td><td>Bio-Rad</td><td>165-2257</td><td>http://www.bio-rad.com</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio</td><td>http://www.zeiss.com/microscopy</td></tr><tr><td>KOD One Step PCR Kit</td><td>EMD Millipore</td><td>71086-4</td><td>http://www.emdmillipore.com</td></tr><tr><td>One Step RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td>www.qiagen.com</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>www.promega.com</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>www.qiagen.com</td></tr><tr><td>Mini Beadbeater - 1</td><td>BioSpecs</td><td>3110BX</td><td>http://www.biospec.com</td></tr><tr><td>Microfuge 18 Centrifuge</td><td>Beckman Coulter</td><td>F241.5P</td><td>www.beckmancoulter.com</td></tr><tr><td>Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH</td><td>www.biotek.com</td></tr></tbody></table>

biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans

basidiyomiset Cryptococcus neoformans, merkezi sinir sisteminin bir invaziv fırsatçı patojen, dünya çapında, dünya çapında yılda fazla 625.000 ölümle sonuçlanan mantar menenjitin en sık nedenidir. Elektroporasyon Cryptococcus plasmidlerin dönüştürülmesi için geliştirilmiş olmasına rağmen, sadece biyolistik homolog rekombinasyon ile genomun içine entegre edilebilir lineer DNA dönüşümü için etkili bir araç sağlar. Asetat HIV enfeksiyonu sırasında büyük bir fermentasyon ürünü olduğu gösterilmiştir, ancak bu önemi henüz bilinmemektedir. Enzimler ksiluloz-5-fosfat / fruktoz-6-fosfat fosfoketolaz (Xfp) ve asetat kinaz (ACK) oluşan bir bakteriyel yol C&#39;de asetat üretimi için üç potansiyel geçiş yollarından biri olup neoformans. Burada, bir yapının Biyolistik dönüşüm gösterir,Ack kodlayan gen, MCherry floresan etiketi kaynaşmış C geri olan neoformans. Daha sonra ACK mahaline içine ACK -mCherry füzyon entegrasyonu onaylayın.

C başarılı bir biolistik transformasyon neoformans bu protokol şeması (Şekil 1) takip edilerek elde edilebilir. DNA, püskürtmenin (Şekil 2A) sonra biyolistik dönüşüm ile kaplanmış altın parçacıkları başarılı bir filiz plaka üzerinde görünür bir altın ile gösterilir. Seçici ortama YPD + 1M sorbitol plakaları kurtarıldı hücreleri kaplama sonra oda sıcaklığında sol Koloniler 4 ila 5 gün içinde görünmelidir. DNA 2 ug Dönüşümü 2...

Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biyolistik dönüşüm homolog yeniden bağlama aracılığıyla fırsatçı patojen Cryptococcus neoformans genomuna DNA&#39;nın kararlı bütünleşmesinin oluşturmak için kullanılan bir yöntemdir. Biz C.neoformans içine floresan etiket mCherry kaynaşmış kodlayan gen asetat kinaz olan bir yapı, biolistik dönüşümü gösterecektir.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

biolistic-transformation-fluorescent-tagged-gene-into-opportunistic

Research

JoVE Journal

Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

9.9K Görüntüleme.  Clemson University.  Biyolistik dönüşüm homolog yeniden bağlama aracılığıyla fırsatçı patojen Cryptococcus neoformans genomuna DNA'nın kararlı bütünleşmesinin oluşturmak için kullanılan bir yöntemdir. Biz C.neoformans içine floresan etiket mCherry kaynaşmış kodlayan gen asetat kinaz olan bir yapı, biolistik dönüşümü gösterecektir. 

Video: Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Floresan protein füzyonları üretilmesi<em&gt; Candida</em&gt; Türler

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Genetik

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bakteriyel gen düzenlemesi enfekte dokularda çalışmaya bir floresan tabanlı yöntemi

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. elegans Excretory kanal hücre içi Lumen morfogenez ve In Vivo polarize membran Biyogenez tek bir hücrede için bir Model olarak: etiketleme GFP-füzyon, RNAi etkileşimi ekran ve görüntüleme

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Gelişim Biyolojisi

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryote genomik Dönüşüm<em&gt; Ostreococcus tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Biyoloji

Chlorella vulgaris'te Agrobacterium tumefaciens Kullanılarak Kararlı Gen Entegrasyonu

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Yazar Spotlight: Chlorella vulgaris için Agrobacterium tumefaciens Kullanılarak Optimize Edilmiş Dönüşüm Protokolü 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Yeşil Mikroalglerin Agrobacterium tumefaciens Aracılı Genetik Mühendisliği ( Chlorella vulgaris)

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Biyomühendislik

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

Candida albicans Cdr1-mGFPHis Analizi için Küçük Ölçekli Plazma Membran Hazırlama

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Filamentöz Cyanobacterium Phormidium lacuna'nın Doğal Transformasyonu, Protein İfadesi ve Kriyokorunumu

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Floresan protein füzyonları üretilmesi

Bakteriyel gen düzenlemesi enfekte dokularda çalışmaya bir floresan tabanlı yöntemi

C. elegans Excretory kanal hücre içi Lumen morfogenez ve In Vivo polarize membran Biyogenez tek bir hücrede için bir Model olarak: etiketleme GFP-füzyon, RNAi etkileşimi ekran ve görüntüleme

Picoeukaryote genomik Dönüşüm

Yeşil Mikroalglerin Agrobacterium tumefaciens Aracılı Genetik Mühendisliği ( Chlorella vulgaris)

Yeşil Mikroalglerin Agrobacterium tumefaciens Aracılı Genetik Mühendisliği ( Chlorella vulgaris)

Yeşil Mikroalglerin Agrobacterium tumefaciens Aracılı Genetik Mühendisliği ( Chlorella vulgaris)

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

Candida albicans Cdr1-mGFPHis Analizi için Küçük Ölçekli Plazma Membran Hazırlama

Filamentöz Cyanobacterium Phormidium lacuna'nın Doğal Transformasyonu, Protein İfadesi ve Kriyokorunumu