DF הוא נתמך על ידי גשרים-NSF המילגה דוקטורט. מיקרוסקופ השתמשו בעבודה זו נתמכת בחלקה על ידי הדמיה מיקרוסקופ רשת תשתית הליבה מענק מקולורדו סטייט. העבודה על מתאמי clathrin במעבדה המחבר הוא נתמך על ידי המז&quot;פ הזנק וקרנות איגוד הלב האמריקני בפרס 09SDG2280525 אל SD

<ol>
	<li>Mellman, I., Warren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+road+taken:+past+and+future+foundations+of+membrane+traffic.">The road taken: past and future foundations of membrane traffic.</a> Cell. 100, 99-112 (2000).</li><li>Engqvist-Goldstein, A. E., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+assembly+and+endocytosis:+from+yeast+to+mammals.">Actin assembly and endocytosis: from yeast to mammals.</a> Annu Rev Cell Dev Biol. 19, 287-332 (2003).</li><li>Conner, S. D., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+portals+of+entry+into+the+cell.">Regulated portals of entry into the cell.</a> Nature. 422, 37-44 (2003).</li><li>Bonifacino, J. S., Traub, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+for+sorting+of+transmembrane+proteins+to+endosomes+and+lysosomes.">Signals for sorting of transmembrane proteins to endosomes and lysosomes.</a> Annu Rev Biochem. 72, 395-447 (2003).</li><li>Ungewickell, E. J., Hinrichsen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis:+clathrin-mediated+membrane+budding.">Endocytosis: clathrin-mediated membrane budding.</a> Curr Opin Cell Biol. 19, 417-425 (2007).</li><li>Doherty, G. J., McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endocytosis.">Mechanisms of endocytosis.</a> Annu Rev Biochem. 78, 857-902 (2009).</li><li>Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin.">Clathrin.</a> Annu Rev Biochem. 69, 699-727 (2000).</li><li>Brodsky, F. M., Chen, C. Y., Knuehl, C., Towler, M. C., Wakeham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+basket+weaving:+formation+and+function+of+clathrin-coated+vesicles.">Biological basket weaving: formation and function of clathrin-coated vesicles.</a> Annu Rev Cell Dev Biol. 17, 517-568 (2001).</li><li>Owen, D. J., Collins, B. M., Evans, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptors+for+clathrin+coats:+structure+and+function.">Adaptors for clathrin coats: structure and function.</a> Annu Rev Cell Dev Biol. 20, 153-191 (2004).</li><li>Dell'Angelica, E. C., Klumperman, J., Stoorvogel, W., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+AP-3+adaptor+complex+with+clathrin.">Association of the AP-3 adaptor complex with clathrin.</a> Science. 280, 431-434 (1998).</li><li>Dell'Angelica, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-binding+proteins:+got+a+motif?+Join+the+network!.">Clathrin-binding proteins: got a motif? Join the network!.</a> Trends Cell Biol. 11, 315-318 (2001).</li><li>Holtzman, D. A., Yang, S., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic-lethal+interactions+identify+two+novel+genes,+SLA1+and+SLA2,+that+control+membrane+cytoskeleton+assembly+in+Saccharomyces+cerevisiae.">Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae.</a> J Cell Biol. 122, 635-644 (1993).</li><li>Howard, J. P., Hutton, J. L., Olson, J. M., Payne, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sla1p+serves+as+the+targeting+signal+recognition+factor+for+NPFX(1,2)D-mediated+endocytosis.">Sla1p serves as the targeting signal recognition factor for NPFX(1,2)D-mediated endocytosis.</a> J. Cell. Biol. 157, 315-326 (2002).</li><li>Mahadev, R. K., Pietro, S. M. D. i., Olson, J. M., Piao, H. L., Payne, G. S., Overduin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Sla1p+homology+domain+1+and+interaction+with+the+NPFxD+endocytic+internalization+motif.">Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif.</a> EMBO J. 26, 1963-1971 (2007).</li><li>Pietro, S. M. D. i., Cascio, D., Feliciano, D., Bowie, J. U., Payne, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+clathrin+adaptor+function+in+endocytosis:+A+novel+role+for+the+SAM+domain.">Regulation of clathrin adaptor function in endocytosis: A novel role for the SAM domain.</a> EMBO J. 29, 1033-1044 (2010).</li><li>Kaksonen, M., Toret, C. P., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+design+for+the+clathrin-+and+actin-mediated+endocytosis+machinery.">A modular design for the clathrin- and actin-mediated endocytosis machinery.</a> Cell. 123, 305-320 (2005).</li><li>Longtine, M. S., McKenzie, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+modules+for+versatile+and+economical+PCR-based+gene+deletion+and+modification+in+Saccharomyces+cerevisiae.">Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.</a> Yeast. 14, 953-9561 (1998).</li><li>Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C., Philippsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+HIS3+marker+and+GFP+reporter+modules+for+PCR-targeting+in+Saccharomyces+cerevisiae.">Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.</a> Yeast. 13, 1065-1075 (1997).</li><li>Robinson, J. S., Klionsky, D. J., Banta, L. M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+sorting+in+Saccharomyces+cerevisiae:+isolation+of+mutants+defective+in+the+delivery+and+processing+of+multiple+vacuolar+hydrolases.">Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.</a> Mol Cell Biol. 8, 4936-4948 (1988).</li><li>Ito, H., Fukuda, Y., Murata, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+intact+yeast+cells+treated+with+alkali+cations.">Transformation of intact yeast cells treated with alkali cations.</a> J. Bacteriology. 153, 163-168 (1983).</li><li>Vida, T. A., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Sikorski, R. S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+of+shuttle+vectors+and+yeast+host+strains+designed+for+efficient+manipulation+of+DNA+in+Saccharomyces+cerevisiae.">A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.</a> Genetics. 122, 19-27 (1989).</li><li>Piao, H. L., Machado, I. M., Payne, G. 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Clathrin מצופה שלפוחית ​​(CCV) להשתתף אנדוציטוזה והובלה של Golgi טרנס רשת endosomes, מסלולים שמורים כי הם בסיסי בביולוגיה של התא האיקריוטים. הגישות המתוארת במאמר זה שיטות מועילים כדי לחקור את המנגנונים המולקולריים האחראים ליצירת CCV, באינטראקציות בין חלבונים מסוימים מתאם cla...

<table border="1" style=";text-align:right;direction:rtl"><th> חומר </th><th> חברה </th><tbody><tr><td> QuickChange-XL אתר מכוונת mutagenesis ערכת </td><td> Stratagene </td></tr><tr><td> מעכב פרוטאז קוקטייל </td><td> סיגמא </td></tr></tbody></table>

in vivo and in vitro studies of adaptor-clathrin interaction

מסלול endocytic הגדולות יוזם עם הקמתה של clathrin מצופה שלפוחית ​​(CCVs) כי מטען הובלה מתא השטח כדי endosomes 1-6. CCVs מאופיינים סריג polyhedral של clathrin קרום כי שלפוחית ​​מעילים משמש פיגום מכני. מעילים Clathrin נאספו במהלך היווצרות שלפוחית ​​מ triskelia clathrin הפרט, את הטופס מסיסים של clathrin מורכבת משלושה כבד ושלושה שרשרת אור יחידות משנה 7,8. מכיוון triskelion אין את היכולת לאגד הממברנה ישירות, clathrin מחייב מתאמים הן קריטיות הקישור סריג clathrin להרכיב את הממברנה דרך בשיתוף עם שומנים ו / או חלבונים בממברנה 9. מתאמי גם הטרנסממברני חבילת מטען חלבון, כגון קולטנים, והוא יכול לתקשר אחד עם השני וגם עם רכיבים אחרים של היווצרות CCV מכונות 9. מעל עשרים מתאמי clathrin תוארו, מספר מעורבים אנדוציטוזה מתווכת clathrin ואחרים למקם את Golgi טרנס רשת או endosomes 9. למעט HIP1R (שמרים Sla2p), כל המתאמים clathrin ידוע להיקשר תחום הטרמינל-N-המדחף של שרשרת כבדה clathrin 9. מתאמי Clathrin הם חלבונים מודולרי המורכב תחומים מקופל המחוברים באמצעות linkers גמישה מובנה. בתוך האזורים הללו והמקשר, מוטיבים מחייב לתווך קצר אינטראקציות עם תחום ה-N-מסוף clathrin או רכיבים אחרים של היווצרות שלפוחית ​​מכונות 9. שני ברורים clathrin מחייב מוטיבים הוגדרו: תיבת clathrin ו-W-9 התיבה. רצף clathrin תיבת קונצנזוס הוגדר במקור L [L / I] [D / E / N] [L / F] [D / E] 10 אבל וריאנטים התגלו לאחר מכן 11. ה-W-box תואמת PWxxW את הרצף (כאשר x הוא שאריות). Sla1p (קטלני סינתטי עם אקטין מחייב חלבון-1) זוהה במקור החלבון אקטין הקשורים הכרחי מבנה נורמלי cytoskeleton אקטין ודינמיקה באתרים endocytic בתאי שמרים 12. Sla1p גם קושר את האות NPFxD מיון endocytic הוא קריטי עבור אנדוציטוזה של מטען הנושאת את האות NPFxD 13,14. לאחרונה, Sla1p הודגם להיקשר clathrin באמצעות מוטיב דומה לתיבת clathrin, LLDLQ, כינה גרסה תיבת clathrin-(VCB), וכן לתפקד מתאם clathrin endocytic 15. בנוסף, Sla1p הפך סמן בשימוש נרחב עבור המעיל endocytic ב לחיות פלואורסצנטי תא מחקרים מיקרוסקופיה 16. כאן אנו משתמשים Sla1p כמודל לתיאור גישות מתאם-clathrin מחקרים אינטראקציה. אנו מתמקדים מיקרוסקופ פלואורסצנטי תא חי, GST, לנתץ, ושיתוף immunoprecipitation שיטות.

Watch this Scientific Journal Video about In vivo and in vitro Studies of Adaptor-clathrin Interaction at JoVE.com

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin בתיווך אנדוציטוזה תלוי חלבונים מתאם לתאם מבחר מטען הרכבה מעיל clathrin. כאן אנו מתארים את נוהלי ללמוד מתאם-clathrin אינטראקציה פיזית הדמיה תא חי באמצעות גישות כמודל מתאם endocytic שמרים Sla1p חלבון.

In vivo ו במבחנה של אינטראקציה מתאם-clathrin

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

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In vivo and in vitro Studies of Adaptor-clathrin Interaction

14.4K Views.  Colorado State University. Clathrin בתיווך אנדוציטוזה תלוי חלבונים מתאם לתאם מבחר מטען הרכבה מעיל clathrin. כאן אנו מתארים את נוהלי ללמוד מתאם-clathrin אינטראקציה פיזית הדמיה תא חי באמצעות גישות כמודל מתאם endocytic שמרים Sla1p חלבון.

Video: In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

במבחנה פליטת אור הדמיה vivo ג&#39;ין הכתב של בתאי גזע עובריים אנושיים

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

ויזואליזציה של תיקון MG53 בתיווך קרום התא באמצעות In vivo ו במבחנה מערכות

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

הבידוד של הריבוזום פוליפפטידים המתהווה כרוכים<em&gt; במבחנה</em&gt; כדי לזהות אתרים השהה של תרגום לאורך ה-mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

גירוי של cytoplasmic מסלולי חישה DNA<em&gt; במבחנה</em&gt; ו<em&gt; בVivo</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

In vivo לחקר האדם האנדותל-pericyte אינטראקציה באמצעות assay Plug מטריקס ג&#39;ל עכבר

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

במבחנה , גישות In Vivo כדי לקבוע חדירות תאים אפיתל המעי

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

במבחנה ניתוח של E3 אוביקוויטין פונקציית ליגאז

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

בידוד תאי שריר חלק לבביים וכלי דם מדגי זברה בוגרים, צעירים, זחליים ועובריים למחקרים אלקטרופיזיולוגיים

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

שיטת צביעה כפולה לזיהוי פקטין באינטראקציה בין צמחים לפטריות

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

ניתוח הסתיידות כלי דם חוץ-תאית בתיווך שלפוחית באמצעות מודלים In Vitro ו-In Vivo

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...