Biz bu iş için fon sağlamak için Ohio State Üniversitesi Kapsamlı Kanser Merkezi&#39;nde minnettarız.

1. Entegre Rekombinasyon Yüzey Hücre Hattı <ol><li> HeLa hücreleri PDR-GFP (M. Jasin, Memorial Sloan-Kettering Kanser Merkezi&#39;nden plazmid hediye 7) ile stabil bir şekilde dönüştürülmüş ve transformants seçimi için puromycin muhafaza edildi. </li><li> Hücreler% 10 fetal sığır serumu, 2 mM GlutaMAX, 1 mM sodyum piruvat, penisilin / streptomisin (100 U / mL/0.1 mg / ml), ve 1.5 ug / ml puromycin içeren DMEM / FBS geçişli. </li></ol> Plazmid ve istikrarlı transformants PDR-GFP rekombinasyon substrat, GFP iki inaktif allel (Şekil 1) içerir. Bir allel I-SCEI 18 bp restriksiyon enzimi tanıma sitesi kodlama sırayla dahil edilmesi nedeniyle devre dışı. Bu hücrelerin I-SCEI İfade çift iplikli DNA sonu (DSB) oluşturur. Bu DNA&#39;nın inaktif GFP ikinci bir allel var, ama ilk allel I-SCEI site ile ilişkili, dizisi kısmını vahşi türüdür. Bu ikinci GFP allel DSB homolog rekombinasyon tamiri için bir dizi donör olarak işlev görebilir ve flow sitometri ile kolayca tespit aktif bir GFP allel neden olacaktır. Alternatif olarak, DSB nonhomologous I-SCEI kısıtlama yok ve GFP-negatif hücreler neden olur, sonunda katılmayı tarafından tamir edilebilir. Bir hücre homolog rekombinasyon GFP pozitif hücrelerin yüzdesi sayarak belirlenir. Bu hücre hattı, pHeLa-DR13-9, sıfır arka plan GFP sinyal ve plazmid, pCBASce ifade I-SCEI transfeksiyon üzerine GFP pozitif hücrelerin nispeten yüksek bir yüzdesi tüm klonlar arasında seçildi. (PCBASce ve vektör kontrol, pCAGGS, M. Jasin Memorial Sloan-Kettering Kanser Merkezi tarafından sağlandı.) HeLa-DR13-9 hücre hattı talebi üzerine yazarları mevcuttur. 2. Transfeksiyon Endojen BRCA1 İncelten ve Geri Mutant BRCA1 ekle. <ol><li> , Ilk transfeksiyon önce (genellikle Salı) günü: 10 cm çanak HeLa-DR13-9 hücreleri en az% 90 konfluent başlayın. </li><li> 1 mL Trypsinize ve 9 mL DMEM / FBS ekleyerek reaksiyonu durdurmak, pipetleme ile iyice karıştırın. </li><li> 40 mcL başına iyi bir 24 plaka 0.5 mL DME / FBS tripsinize hücreleri ekleyin. </li><li> Gün 1 transfections (Çarşamba): daha az baştan başlamak hücreleri, ~% 40-50 birleşmiş olmalıdır. Transfect karıştırılarak: 5 pmol siRNA + 12.5 mcL Opti-MEM 0.3 ug plazmid DNA ifade BRCA1-mutant ve 12.5 mcL Opti-MEM 0.5 mcL Lipofectamine 2000. Lipofectamine protokolü (Invitrogen) göre transfeksiyon ile devam edin. </li><li> Medya 4-6 saat sonra değiştirin. BRCA1 tüketmek için kullandığınız siRNA belirterek BRCA1 3&#39;-UTR dizileri 80.780 - 80.798 (katılım sayısı AY273801) dizisi GCUCCUCUCACUCUUCAGU dayanmaktadır. </li><li> 2. Gün: 6 plaka, 24 plaka trypsinizing hücrelere aktarın. </li><li> 3. Gün transfections: 25 pmol siRNA + 0.75 ug plazmid DNA ifade BRCA1 mutant 62.5 mcL Opti-MEM ve 62.5 mcL Opti-MEM 2.5 mcL Lipofectamine 2000 + 0.75 ug pCBASce (veya pCAGGS vektör kontrolü). Medya 4-6 saat sonra değiştirin. </li></ol> 3. Transfektanları, analizi. <ol><li> 6 gün, her bir kuyudan hücreleri trypsinize ve 1 ml DMEM / FBS durdurmak. GFP ifade hücreleri incelendiğinde, 5 veya 7 gün yapılabilir, ama biz 6 eserleri o günün en iyi bulmak olabilir. </li><li> Flow sitometri ile ortalama 10.000 hücre analiz edin. Ohio State Üniversitesi Kapsamlı Kanser Merkezi Analitik Sitometri paylaşılan kaynak Becton Dickinson FACSCalibur enstrüman kullanın. Tek, canlı hücreleri ileri ve yan dağılım parametreleri kullanarak kapılı. GFP pozitif hücrelerin GFP floresan dedektör (FL1) kullanılarak tespit edilir ve sonuçları bir histogram (Şekil 2, alt) olarak sunulmuştur. </li><li> Sol içinde hücreler, istenirse floresan mikroskobu fotoğrafçılığı için replated olabilir. </li></ol> 4. Temsilcisi Sonuçlar: BRCA1 homolog rekombinasyon 6,11 çift zincirli DNA kırıkları tamir için yolu düzenler. Homolog rekombinasyon onarım geçiren bu hücrelerin kolayca floresan mikroskobu (Şekil 2, üst) tarafından tespit edilir. BRCA1 sonuçları tespit GFP pozitif hücreler (Şekil 2, sağ) bir azalma tükenmesi. FACSCalibur çıktı, X ekseni ve Y ekseni (Şekil 2, alt) hücrelerinin sayısı, hücre başına GFP yoğunluğu ile bir histogram. Tipik bir tek bir deney sonuçları seti, hücrelerin% 15.5 I-SCEI ifade ve kontrol RNAi tükenmesi (Şekil 3) sonra GFP pozitif. Buna karşılık, BRCA1 ve vektör tükenmesi% 0.7 geri GFP-dönüşüm azalır ekleyin. Add-back yabani tip BRCA 1 veya BRCA 1 (I21V) tamamen restore homolog rekombinasyon,% 15-16 oranında, GFP-pozitif hücrelerin elde algılandı. BRCA1 (M1 add-back8T) mutant (Şekil 3, şerit 5), vektör kontrol add-back olarak benzer bir etkisi vardı. Bu varyant 9 endojen protein olarak benzer seviyelerde ifade beri, biz böylece BRCA1 (M18T) varyant homolog rekombinasyon fonksiyonel olmayan yorumlamak. Biz genellikle I-SCEI ifade plazmid transfecting sonra GFP pozitif dönüştürmek hücrelerin% 10-20 var. BRCA1, tükenmesi tekrarlanabilir, yaklaşık 8-10 kat GFP pozitif hücrelerin dönüşüm azaltır. GFP pozitif hücreler gerçek yüzdeleri, bir deney içinde kontrol tükenmesi göre BRCA1 tükenmesi deney deney, GFP pozitif hücrelerin oranı değişebilir rağmen oldukça tutarlıdır. Biz sınır tanımayan dönüşüm oranı% 100 ve yüzde olarak BRCA1 tükenişleri ve eklenti sırtlarını ifade sonuçları ayarlayarak sonuçları normalleştirmek. Bu şekilde, birden fazla deneyler birleştirmek ve deneysel bir varyasyon yüksek değildir. Şimdiye kadar sağlam sonuçlar vermiştir BRCA1 mutantlar geri ekleyin: her varyant BRCA1 ya tam homolog rekombinasyon aktivite geri ya da herhangi bir etkisi olmuştur. Bu sonuçların elde edilmesinde önemli bir göz transfeksiyon koşulları tedavi edilmezse hücrelerinde endojen protein konsantrasyonu yaklaşık eşit BRCA1 protein seviyeleri üretmek. BRCA1 bile kusurlu bir mutant bir aşırı ekspresyonu çok yüksek bazı olumlu homolog rekombinasyon aktivite neden olabilir. Üstesinden gelmek için gerekli bir sorun transfeksiyon toksisitesi oldu. BRCA1 kendisi tükenmesi yavaş hücre büyümesi ve çanak hücrelerinin sayısına göre Lipofectamine-2000 ve plazmid DNA dengesi, tutarlı bir düzeyde tutulmalıdır. Çok fazla Lipofectamine veya plazmid DNA, hücre sayısını azaltmak ve hücrelerin sayısı, güvenilir flow sitometri sonuçları elde etmek için yetersiz olacaktır. Transfekte hücrelerinin sayısı karşısında plazmid ve Lipofectamine reaktiflerin denge hücreleri canlılığını korumak için oldukça önemlidir. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Şekil 1" /> Şekil 1 rekombinasyon substrat. M. Jasin 7,12 grubu tarafından geliştirilen strateji tasvir edilmiştir. PDR-GFP plazmid I-SCEI kısıtlama endonükleaz site içermektedir biri GFP iki hatalı allel içerir. Bir HeLa kaynaklı hücre hattı, bu DNA, tek bir genom 9 yerinde entegre substrat ve aktif onarım, GFP-pozitif bir allel dönüşüm homolog rekombinasyon sonuçları I-SCEI yerinde çift iplikli DNA sonu içerir. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Şekil 2" /> Şekil 2 floresan mikroskopi GFP pozitif hücrelerin tespiti. Hücreler bu protokol uyarınca, test ve kontrol tükenmiş hücreleri GFP ifade (solda) ile hücreler gibi yüksek bir oranda tespit, homolog rekombinasyon aktif. GFP pozitif hücreler (sağda) sayısında keskin bir düşüş RNAi sonuçlarına göre BRCA1 tükenmesi. <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Şekil 3" /> Şekil 3 BRCA1 missense mutantlar bir add-back Sonuçları. Bu histogramda tek bir deney sonuçları gösterilmiştir. Plazmid ifade transfekte I-SCEI yokluğu (şeritli 1) tespit hiçbir GFP pozitif hücreler vardır. Plazmid ifade I-SCEI 3. gününde transfekte edildiği hücrelerde şerit 2-6 sonuçlar alınmıştır. Ilgisiz bir gen (lusiferaz) tükenmiş ve vektör add-back, hücrelerin% 15.5 (şeritli 2), GFP pozitif hücreler. BRCA1 ve vektör tükenmesi add-back homolog rekombinasyon BRCA1 kaybı etkisi (şeritli 3) ortaya koymaktadır. BRCA1 tükenmesi ve yabani tip BRCA1 (şerit 4), BRCA1 (M18T) (şeritli 5), BRCA1 (I21V) (şeritli 6) add-back etkileri gösterilmiştir. Bu sonuçlar, tek bir deney, homolog rekombinasyon, belirli bir mutant BRCA1 protein tarafından desteklenen bir önlem alabilmek için en az iki veya daha fazla tekrar deneyleri ile birlikte olacaktır.

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	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. 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Bu yöntemin avantajı, yabani tip endojen ifade BRCA1 hücreleri kullanılarak homolog rekombinasyon DNA onarımı düzenleyen BRCA1 protein fonksiyonu çalışma. Biz iki ayrı kurulmuş yöntemleri homolog rekombinasyon sürecinin eğitim ve DNA tamir fonksiyonu için BRCA1 varyantları test etmek için yeni bir yöntem elde etmek için RNAi-aracılı tükenmesi kullanan kabul ettiler. Bu yöntemin başarısı için anahtar GFP dönüşüm çok yüksek düzeyde elde böyle genom rekombinasyon substrat gibi HeLa gibi kolay transfectable bir hücre hattı kurmak oldu. Saptanabilir arka plan GFP sinyali elde etmek için birden fazla klonlar orijinal dönüşüm elenmiş, ama plazmid I-SCEI ifade transfeksiyon üzerine GFP dönüşüm çok yüksek bir düzeyi vardı. Bu yöntem ile, şimdi homolog rekombinasyon fonksiyonu için diğer özel BRCA1 amino asit kalıntıları araştırıyoruz. Bu çalışma, biyolojik anlayışlar ile birlikte, bu sonuçlara klinik kanser genetiği uygulanabilir. BRCA1 missense mutasyon yüzdesi yüksek, çok nadir varyantları vardır beri bilinmeyen klinik önemi vardır ve bu çok genetik segregasyon analizi 3,10,13,14 kanseri ilişkiyi belirlemek için yeterli bilgi yoktur . Bu yöntemi kullanarak, BRCA1 homolog rekombinasyon düzenleyen biyolojik işlevi belirli bir varyant sonuçlarını kendi genomunda bu varyantlarının bir taşıyan kadınları bilgilendirmek için kullanılabilir.

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<td>Invitrogen</td>
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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

Missense mutasyon, fonksiyonel analiz endojen protein hücre varlığı ile karmaşık olabilir. BRCA1 yapı-işlev analizleri, tam uzunlukta BRCA1 protein ve hypomorphic BRCA1 protein 1,2,3,4,5 ifade hücre hatları ile çalışan doğasında zorluklar için sağlam bir tahlil eksikliği ile komplike olmuştur . Biz bir hücreye endojen BRCA1 protein akut BRCA1 mRNA 3&#39;-UTR hedefleyen RNAi tükenmiş ve bir plazmid BRCA1 varyant ifade co-transfecting yerini sayede bir sistem geliştirdi. Bu prosedürün bir avantajı BRCA1 ve eş zamanlı yedek akut susturulması hücreleri BRCA1 fonksiyon kaybı telafi etmek için zaman içinde ortaya çıkabilecek ikincil mutasyonlar veya uyarlamalar olmadan büyümeye izin veren. Bu tükenmesi ve add-back işlemi kolayca homolog rekombinasyon aktivite için test HeLa kaynaklı hücre hattı yapıldı. Homolog rekombinasyon tahlil rekombinasyon substrat genom (Şekil 1) 6,7,8,9 entegre sayede daha önce yayınlanmış bir yöntem dayanmaktadır . Bu rekombinasyon substrat etkin olmayan bir GFP allel içinde nadir kesim I-SCEI restriksiyon enzimi sitesi ve aşağı ikinci bir inaktif GFP allel. Homolog rekombinasyon tarafından tamir edilebilir çift iplikli bir ara, I-SCEI sonuçları ifade ve homolog rekombinasyon ara tamir eğer kolayca GFP protein ekspresyonu için flow sitometri tarafından atılırsa aktif bir GFP allel oluşturur plazmid, Transfeksiyon . Endojen BRCA1 tükenmesi homolog rekombinasyon aktivite 8-10 kat azalma ve vahşi-tip plazmid tam homolog rekombinasyon fonksiyonu restore add-back. Belirli bir noktaya tam uzunlukta BRCA1 mutantlar co-transfekte plazmid ifade edildi, özel missense mutant etkisi atılır. Bir örnek olarak, BRCA1 (M18T) protein, bilinmeyen bir klinik önemi 10 varyant, ifade, bu hücrelerin de ifade edildi, homolog rekombinasyon BRCA1-bağımlı geri yüklemek için başarısız oldu . Bilinmeyen bir önemi de başka bir versiyonudur kontrast, ifade, BRCA1 (I21V) tam BRCA1 bağımlı homolog rekombinasyon fonksiyonu restore. BRCA1 missense mutasyon fonksiyon testleri Bu strateji centrosome fonksiyonu (Kais ve ark, yayınlanmamış gözlemler) assaying başka bir biyolojik sistemi tatbik edilmiştir. Genel olarak, bu yaklaşım, resesif analiz edilmelidir herhangi bir gen missense mutantların analizi için uygundur.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Biz RNAi kullanarak bir hücrenin endojen BRCA1 protein tüketen ve bir kodlama değişiklik içeren bir BRCA1 noktası mutant yerine homolog rekombinasyon DNA hasarı tamir için bir doku kültürü bazlı analiz BRCA1 varyantları test etmek için bir yöntem sağlar.

Endojen Wild-tip BRCA1 Express hücreler kullanarak Homolog Rekombinasyon BRCA1 Mutasyonlar Etkileri belirlenmesi

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

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14.5K Görüntüleme.  The Ohio State University. Biz RNAi kullanarak bir hücrenin endojen BRCA1 protein tüketen ve bir kodlama değişiklik içeren bir BRCA1 noktası mutant yerine homolog rekombinasyon DNA hasarı tamir için bir doku kültürü bazlı analiz BRCA1 varyantları test etmek için bir yöntem sağlar.

Video: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Matrix Rijitlik Akrilamid tabanlı Hidrojeller kullanarak Hücresel Fonksiyon Üzerindeki Etkilerinin incelenmesi

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biyoloji

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Polarize Epitel Hücreleri içine Plazmidler, Mikroenjeksiyon Küçük GTPases Fonksiyon Analizi

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-tip ve Süs Çeşitler arasında ayırt A PCR tabanlı Genotiplendirme Yöntemi Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Homolog rekombinasyon Oluşumlar Recombineering<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Üzerinde Bakterilerin Etkilerinin Ölçme<em&gt; C. Elegans</emBir Yumurta Tutma Testi Kullanma&gt; Davranış

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

Whole Sırasının NGS Analiz için bir transpozaz tabanlı Technology tarafından gDNA Zenginleştirme<em&gt; BRCA1</em&gt;<em&gt; BRCA2</emDNA Hasarı Onarım ilgilendim&gt; ve 9 Genler

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Peptit Diziler protein-protein etkileşim Siteleri belirlenmesi

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Karaciğer ve Süt örneklerinde vahşi tipli ve sialik asitler tayini ve<em&gt; CMAH</em&gt; Knock-out Fare.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Bakteriyosin Etkilerini Farenin Bağırsak Mikrobiyolojisi Üzerine Etkileyen Bir Yöntem

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Zararlı etkileri, düşük basınç plazma sterilizasyon Bacillus subtilis sporlar kullanarak canlı cep mikroskobu sağkalıma soruşturma

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...