Finansman NIH / NIAID hibe RO1-AI-63457 ve arra hibesine ek RO1-AI-63457 S1 tarafından sağlanmıştır. Biz laboratuvar grubu ve işbirlikçilerinin tüm mevcut ve geçmiş üyeleri katkıları kabul etmiş sayılırsınız.

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+leprosy+for+control+programmes.">Chemotherapy of leprosy for control programmes.</a> Technical Report Series. 675, 18-22 (1982).</li><li>Cole, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+gene+decay+in+the+leprosy+bacillus.">Massive gene decay in the leprosy bacillus.</a> Nature. 409, 1007-1011 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leproma+Web+Server.">Leproma Web Server.</a> Leproma Web Server [Internet]. , (2004).</li><li>Groathouse, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Polymorphic+Loci+for+Molecular+Typing+of+Strains+of+Mycobacterium+leprae.">Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium leprae.</a> J. of Clinical Microbiology. 42, 1666-1672 (2004).</li><li>Young, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Short+Tandem+Repeat+Sequences+to+Study+Mycobacterium+leprae+in+Leprosy+Patients+in+Malawi+and+India.">Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India.</a> Public Library of Science: Neglected Tropical Diseases. 2, E214-E214 (2008).</li><li>Monot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Variable-Number+Tandem+Repeats+Appropriate+for+Genotyping+Mycobacterium+leprae.">Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae.</a> J. of Clinical Microbiology. 46, 2291-2297 (2008).</li><li>Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Variable+Number+Tandem-Repeat+Genotyping+for+Mycobacterium+leprae+Clinical+Specimens.">Rapid Variable Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens.</a> J. of Clinical Microbiology. 47, 1757-1766 (2009).</li><li>Weng, X. i. a. o. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Distribution+of+Mycobacterium+leprae+Genotypes+in+a+Region+of+High+Leprosy+Prevalence+in+China:+a+3-Year+Molecular+Epidemiological+Study.">Identification and Distribution of Mycobacterium leprae Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study.</a> J. of Clinical Microbiology. 45, 1728-1734 (2007).</li><li>Weng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+leprosy+in+Qiubei+County,+Yunnan,+China:+Insights+from+an+eight+year+molecular+epidemiology+investigation.">Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an eight year molecular epidemiology investigation.</a> Infection, Genetics and Evolution. , (2010).</li><li>Sakamuri, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-Based+Molecular+Epidemiology+of+Leprosy+in+Cebu,+Philippines.">Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines.</a> J. of Clinical Microbiology. 47, 2844-2854 (2009).</li><li>Fontes, A. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+Mycobacterium+leprae+isolates+from+Brazilian+leprosy+patients.">Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients.</a> Leprosy Review. 80, 302-315 (2009).</li></ol>

Lepra hastalardan alınan deri örneklerinin toplanması cilt kliniklerinde çalışan yetenekli klinisyenler veya teknisyenler gerektirir. Bu örnekleri ele Laboratuar çalışanları, laboratuvar mont, eldiven ve koruyucu gözlük takın ve enfekte insan veya armadillo doku örnekleri ele alırken bir biyo-güvenlik kabini içinde çalışmak çok dikkat etmelidir. Dezenfeksiyon yüzeyler ve araçları da önemlidir. DNA örnekleri kirlenmesini önlemek için temiz, steril bir biyo-güvenli kabine çalışmak önemlidir. DNA ekstraksiyonu Qiagen gibi şirketlerden ekstraksiyon kitleri geliştirme sayesinde nispeten kolay hale gelmiştir. Yol dikkatle takip edilmelidir. Kullanılmadığı zaman Tüm numuneler soğuk tutulmalıdır. Örnekleri için tekrarlanan, aşırı sıcaklık değişimleri kaçının. Bu çalışmada kullanılan DNA primerleri Bu yazının sonunda literatür referansları listesinde atıf almıştır çeşitli şirketlerden sipariş edilebilir. Kontaminasyonu önlemek için primerler ile DNA özgür bir ortamda çalışması için önlemler alınmalıdır. Astarlar kuru toz olarak gelir ve TE ile karıştırılır ve bir 100μM konsantrasyona seyreltilmelidir, sonra da küçük miktarlarda (Şekil 2a) ayrılır . Astar Çalışma çözümleri 10μM konsantrasyonları daha seyreltilir. Yine, alanları / primerler kullanılır ve hazırlanan davlumbaz DNA örnekleri kullanmayın. PCR ürünleri kullanılmadığı zaman da soğuk muhafaza edilmelidir. % 3 agaroz jel hazırlanması daha iyi DNA bant ayrımı vermek, ama çalıştırmak için uzun sürer. Tamamen niteliksel amaçlar için,% 2 genellikle yeterlidir. Agaroz jel DNA boyama için kullanılan etidyum bromür çözüm son derece aktif genotoxin deri yoluyla emilir. Bu çözüm ve büyük bir özenle ve eldiven giymek için her zaman emin olmak boyanmış jeller taşıyınız. Herhangi bir DNA numune veya etidyum bromür malzemeleri dokunduktan sonra ellerinizi iyice yıkayın. Etidyum bromür boyama çözüm atın, kurumsal kurallarına göre yıkama ve jeller. Jeller rutin olarak gerekli değildir; FLA yöntemleri kurulduktan sonra bu adımı ortadan kaldırılabilir. Zaman ve reaktif alıcıdır. FLA için numune hazırlamada kullanılan formamid çözüm de son derece toksik ve dikkatle ele alınmalıdır. Kendi kullanımdan sonra ellerinizi yıkayınız. Kurumsal yönergeleri izleyerek atın. Tepe Tarayıcı yazılımı kullanarak FLA sonuçları okunması zor olabilir. Allel aramaları (tandem tekrarlar sayısı) bir temel set CSU (Tablo 4-7) geliştirilmiştir. (M. diğer suşları leprae çalışılmaktadır 4-7 Tablolar herşey dahil olmayabilir dikkat edilmelidir. Listelenen aralıkları dışında kopya sayıları ile allel olabilir.) Bir nevi meydan okumadır &#39;kekeme&#39; doruklarına içerir. Bu aileler, özellikle sadece böyle 10 (TA) 2-3 baz çifti tekrarlar içeren Şüpheli İşlem zirvelerinden. Bazen okumak için doğru tepe seçmek zordur (Şekil 7). Bu şekilde, 190 bp pozitif kontrol tepe ana zirve. 2 yüksekliği düşük Peaks, 4, hatta 6 baz çiftinden daha büyük veya daha küçük ana zirve &#39;kekeme doruklarına.&#39; Denir A-atık da karışıklığa neden olabilir. A-atık önceki metin ve Şekil 8&#39;de açıklanmıştır. Son olarak, PCR ürünlerinin örnekleri oldukça bol, zirveleri çift sivri görünebilir. Bu durumda, tepe formu merkezi okuyun. (Bkz: Şekil 6, Hasta 6 ) Veri yönetimi, bu tür çalışma için zorlu bir görev olabilir. Bir görev ya da işlem tarihinden, operatör, şerit / plaka haritalar, FLA siparişleri, PCR koşulları ve yemek tarifleri, jeller ve fotoğraflar tarihleri, depolama sıcaklıkları, DNA örneğinin dilüsyonları, FLA: tüm ilgili gibi tüm deneyler için bilgi kaydetmek için önemlidir. elektronik dosya depolama yerleri, vb İyi organizasyon ve veri yönetimi, gelecekte belirli bilgi parçaları için harcanan zaman saatlerce kaydedebilirsiniz. Burada açıklanan laboratuar çalışması, Colorado State Üniversitesi ve dünyadaki diğer yerlerde birkaç yıldır devam etmektedir. Toplanan verilerin her türlü vasıta ve ileride kullanmak nasıl olabilir ne büyük resim ortaya çıkmaya başlıyor. Şekiller 10A ve 10B bu DNA parmak izi farklı M. ayırt etmek için nasıl kullanılabileceğini göstermek ülkelerde (10A) ve hatta arasında aile (10B) leprae suşları arasında. Aile ve toplum bağlantılı durumlarda M. taşımak için gösterilmiştir. leprae benzer veya aynı VNTR zorlanma türleri. Umut iletim şebekeleri erken bir algılama sistemi, bu insanlar ecek için geliştirilmiş olabilir cüzam iletim modu (ler) böylece daha fazla fikir edinmek mümkün olabilir.t risk ve tedavi edici ilaç tedavisi, kalıcı nörolojik ve dermatolojik hasar yapılmadan önce başlayabilir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> DNeasy Kan ve Doku Seti </td><td> Qiagen </td><td> 69504 </td><td></td></tr><tr><td> Multiplex PCR seti </td><td> Qiagen </td><td> 206143 </td><td></td></tr><tr><td> DNA Astarlar </td><td> Çeşitli </td><td></td><td></td></tr><tr><td> Agaroz Jel </td><td> Çeşitli </td><td></td><td></td></tr><tr><td> 5x veya 6x Jel Yükleniyor Tampon </td><td> New England Biolabs </td><td> B7021S </td><td> Kendi * yapmak için kullanılabilir Tarifler </td></tr><tr><td> Ethidium Bromür çözümü </td><td> Çeşitli </td><td></td><td> Con ile seyreltilir. </td></tr><tr><td> Hi-Di Formamide Çözüm </td><td> Applied Biosystems </td><td> 4311320 </td><td></td></tr><tr><td> Gene Tarama -500 Liz </td><td> Applied Biosystems </td><td> 4322682 </td><td></td></tr></tbody></table> <a href="http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html">* Http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html</a> <table border="1"><tbody><tr><th> Ekipman </th><th> Yorumlar </th></tr><tr><td> 2 Buzdolapları </td><td> 4 ° C: DNA örnekleri için 1, 1, astar ve Multiplex reaktifler </td></tr><tr><td> 1 adet mikrodalga fırın </td><td> Agaroz ve su jeller yapmak için ısıtma veya uygun bir şekilde </td></tr><tr><td> 1 otoklav </td><td> Malzemelerin sterilizasyon veya düdüklü tencere </td></tr><tr><td> PCR makinesi </td><td> PCR </td></tr><tr><td> 2 takım pipet </td><td> 0.5-10 ul, 10-100 ul, 20-200 ul, astarlar için 1000 ul 1 set, 1 set DNA </td></tr><tr><td> Denizaltı jel elektroforezi kutusu </td><td> Formlar hazırlanması jeller ve taraklar. </td></tr><tr><td> Elektroforez Güç kaynağı </td><td> Jel kutusu bağlantı kabloları ile </td></tr><tr><td> Isı blok </td><td> Eppendorf tüpleri için </td></tr><tr><td> Santrifüj </td><td> Eppendorf tüpleri / şeritler için </td></tr><tr><td> Kapiler elektroforez sistemi (genetik analizör) </td><td> Veya bu analiz, bir kurumun erişim </td></tr><tr><td> Plastik </td><td> Tek kullanımlık aerosol pipet uçları, Eppendorf tüpleri, 8-iyi şeritler, 96-iyi plakaları, optik kalitesi </td></tr><tr><td> İnternet bağlantısı olan bilgisayar </td><td></td></tr></tbody></table>

dna fingerprinting of mycobacterium leprae strains using variable number tandem repeat (vntr) - fragment length analysis (fla)

Cüzam iletim çalışma etkeni bu yana, Mycobacterium leprae, laboratuvar ortamında kültüre edemez özellikle zordur . Bakteriler tek kaynakları cüzzam hastaları ve deneysel olarak enfekte armadillos ve çıplak farelerin. Böylece, modern epidemiyolojisi kullanılan yöntemlerden birçok cüzam çalışma mevcut değildir. Lepra için DSÖ 1 tarafından uygulanan kapsamlı bir küresel uyuşturucu tedavi programına rağmen, cüzzam, yaklaşık 250.000 yeni vaka, her yıl pek çok ülkede endemik olarak kalır. 2 tüm M. leprae (mikro-ve minisatellites olarak da adlandırılır) genom 3,4 eşlenen olmuştur ve birçok lokusların 2 veya daha fazla baz çiftinden segmentleri tekrarlanan olduğu tespit edilmiştir. M. 5 Klinik suşları leprae bu lokuslar birçok tandem tekrarlanan segmentlerinin sayısı (kısa tandem tekrarlar, STR) değişebilir. 5,6,7 Değişken numarası tandem repeat (VNTR) 5 analizi, cüzzam basilinin farklı suşları ayırt etmek için kullanılır olmuştur . Lokusların bazıları diğerlerine göre daha istikrarlı, diğerleri aynı hasta, bazen daha hızlı bir şekilde değiştirmek gibi görünüyor ise, tekrar sayısı daha az varyasyon gösteren görünür. Belirli VNTRs değişkenliği gerginlik yazarak 7,8,9 için uygunluğuna ilişkin sorular getirdi, ortaya çıkan veriler, istikrar içinde farklı birden fazla lokusların, analiz etmek, değerli bir epidemiyolojik araç olarak kullanılabileceğini göstermektedir. Birden fazla lokus VNTR analizi (MLVA) 10 Çin 11,12, Malavi 8, Filipinler 10,13, 14 ve Brezilya da dahil olmak üzere birçok ülkede cüzzam evrim ve iletim incelemek için kullanılır olmuştur. MLVA birden fazla adım içerir. İlk olarak, bakteriyel DNA klinik biyopsilerin veya yarık cilt smear (SSS) konak doku DNA ile birlikte ayıklanır 10 istenilen lokusların sonra çıkarılan DNA polimeraz zincir reaksiyonu (PCR) ile güçlendirilir. 4-5 farklı lokusların için floresan etiketli primerler sonra istenen DNA segmentleri varlığını doğrulamak için 10 PCR ürünleri agaroz jel elektroforezi tabi olabilir. Dört reaksiyon toplam 18 lokusların amplifiye olan, reaksiyon başına kullanılan ve kapiller elektroforez kullanarak floresan parçası uzunluğu analizi (FLA) için sunulmuştur. Armadillo gelen DNA pozitif bir kontrol olarak kullanılan her bir lokus için tekrar kopya sayısı ile bilinen bir bakteri geçişli. FLA kromatogramlar sonra Tepe Tarayıcı yazılımı ve parça uzunluğu VNTR kopya (alleli) sayısı dönüştürülür kullanılarak incelenmektedir. Son olarak, VNTR haplotipleri kalıpları için analiz edilir ve hastanın klinik veriler ile bir araya geldiğinde gerginlik türlerinin dağılımını izlemek için kullanılabilir.

Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Neden Lepra<emLeprae&gt; Mycobacterium</em&gt;, Birçok yerde hala endemiktir. Yayılması ve cüzam iletim modu hakkında bilgi edinmek için, bunu belirlemek için önemlidir suşu<em&gt; M. leprae</em&gt; Bir hasta enfekte. Değişken sayıda tandem tekrarlar (VNTR) yazarak, böyle bir yöntem.

DNA parmak izi<emLeprae> Mycobacterium</emFragment Lenght Analizi (FLA) - Değişken Numarası Tandem Repeat (VNTR)> Suşlarının

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory.  ...

dna-fingerprinting-mycobacterium-leprae-strains-using-variable-number

Research

JoVE Journal

Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

26.9K Görüntüleme.  Colorado State University. Neden Lepra Mycobacterium, Birçok yerde hala endemiktir. Yayılması ve cüzam iletim modu hakkında bilgi edinmek için, bunu belirlemek için önemlidir suşu M. leprae Bir hasta enfekte. Değişken sayıda tandem tekrarlar (VNTR) yazarak, böyle bir yöntem.

Video: DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Tek iplikli DNA, çift iplikli DNA ve RNA Hidroksiapatit Kromatografisi kullanarak bir Çevre Viral Topluluk ayrılması

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

İmmünoloji ve Enfeksiyon

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Büyüme Mycobacterium tuberculosis Biyofilmler

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Genetik Manipülasyon<em&gt; Δku80</emFonksiyonel Genomik Analiz&gt; Suşlarının<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

Için optimize Protokoller<em&gt; Mycobacterium leprae</em&gt; Gerilme Yönetimi: Atimik Çıplak Farelerde Dondurulmuş Hazır Koruma ve Bakım

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Bir yoğun Transposon ekleme kütüphane kullanarak bakteriyel konjugasyon Escherichia Coli Enterobacterial suşları gibi veya Shigella flexneri oluşturulması

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analiz 18FDG PET/BT uzmanı Mycobacterium tüberküloz enfeksiyon ve insan olmayan primatlarda tedavisi için bir araç olarak görüntüleme

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

DNA parmak izi Mycobacterium Suşlarının