עבודה זו נתמכה על ידי הקואליציה סרטן גאורגיה מענק-R9028.

<ol>
	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, 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=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

הייצור וידאו עבור מאמר זה נערך בחסות תרמו פישר סיינטיפיק, אשר מייצרת ריאגנטים מכשיר בשימוש בפרסום זה.

העובדה RNA יכולים להעביר מידע גנטי ישירות הדנ&quot;א הגנומי בתאים התגלה ניצול השימוש RNA המכיל oligos סינתטי (oligos Dharmacon מסונתז) 1. זה היה הוכחה של העיקרון כי ניתן להשתמש בתאים המכילים מולקולות RNA או RNA בלבד רצפים כתבניות עבור סינתזה של DNA. השימוש RNA המכיל oligos לא רק הוביל להפגנה כי המידע הגנטי ניתן להעביר ישירות מ-RNA ל-DNA הגנומי ללא צורך הפוכה עיבד DNA להעתיק ביניים, אלא גם הוכחה כי RNA יכול לשמש כתבנית הומולוגיים ב את התיקון של נזק לדנ&quot;א 1,3. Oligos יכול להתבצע של RNA בלבד או להכיל רק ribonucleotide בודד מוטבע ברצף ה-DNA, כמו בדוגמה שהצגנו כאן. RNA המכיל אוליגו יש אחד ribonucleotide מוטבע שנועדה לתקן את המוטציה שטויות את האלל גנומית trp5 פגום. העברת מידע גנטי ribonucleotide ל-DNA הגנומי מתגלה על ידי שינוי פנוטיפ (תא גדל על המדיום חסר טריפטופן) של התאים הממוקדים. התדירות של תיקון הגן שהושגו עם אוליגו RNA המכיל נמדד בהשוואה לזה של ה-DNA רק לשלוט אוליגו. על ידי ביצוע תיקון זה גן assay ברקע תאים שונים, כאשר אנו עוברים מוטציה גנים שמרים שונים, אנו יכולים לזהות מה גורם / ים משפיעים במיוחד על מיקוד על ידי RNA המכיל oligos. לפיכך, אנו יכולים לחשוף מנגנונים כיצד תאים מווסתים את היציבות של RNA / DNA כלאיים. פשוט על ידי תכנון גרסאות שונות של RNA המכיל oligos אנו יכולים לקבוע את הסבירות עבור מערכת RNA מסוים לשמש תבנית שינוי ה-DNA, ואנחנו יכולים לקבוע את יציבות מסוימת רצפי הרנ&quot;א מוטבע לתוך ה-DNA. יתר על כן, אנו יכולים לזהות מה הם העדיפו בשנת מצעים vivo גורמים אינטראקציה עם RNA / DNA כלאיים. בעוד מספר מחקרים במבחנה, בעיקר ניצול קצר RNA המכיל oligos, נערכו כדי לאפיין את הפונקציה של גורמים שיכולים לזהות רנ&quot;א היברידית עם ה-DNA, כגון אנזימים RNase H 4, בפונקציות vivo של RNases H כמו גם כזהות של חלבונים אחרים שיכולים להשפיע על RNA / DNA יציבות היברידית אינם ידועים ברובם. האפשרות לנצל RNA המכיל oligos באורך משמעותי (50-80, מרס) ובאיכות אופטימלית (כגון Thermo Scientific Dharmacon RNA המכיל oligos) יש לפתוח את הדרך בחינת מגוון רחב של תהליכים מולקולריים ישירות in vivo ב תאים של עניין. כפי שניתן לראות השינוי הזה וידאו, באמצעות RNA המכיל oligos דורש בעצם רק כמה צעדים נוספים לעומת השינוי באמצעות מולקולות ה-DNA, כדי למנוע השפלה ידי RNases. לכן, השינוי באמצעות RNA המכיל oligos אינו מוגבל למערכת שמרים, אבל יכול להיות מיושם על כל אורגניזם או סוג התא שבו שינוי ה-DNA על ידי oligos הוא מיומן. לסיכום, על ידי מיקוד תאים המכילים RNA oligos מספקת את ההזדמנות כדי לייצר RNA / DNA היברידיים ribonucleotides מוטבע ב DNA בתאי in vivo. היציבות, פונקצית וההשלכות של אלה in vivo שנוצר RNA / DNA כלאיים ניתן לנתח ומאופיינת, פוטנציאל חשיפת מנגנוני ידוע של תיקון DNA וגילוי אסטרטגיות חדשניות גן מיקוד.

א טרנספורמציה ריאגנטים המדיה (שונה מן Storici ו רזניק, 2006 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> YPD (שמרים Peptone דקסטרוז): עבור L 1, 10 גרם תמצית שמרים, 20 גרם סויה peptone, 20 גרם דקסטרוז. הוסף 15 גר &#39;אגר לעשות התקשורת מוצק YPD. החיטוי לפני השימוש. אחסן בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> פתרון 1: 0.1 M של ליתיום אצטט. הכן מיד לפני השינוי. פתרון 1 הוא פתרון של העבודה, ולכן הוא מוכן ישירות האבקה. פתרון ללא מניות הוא עשה. לשמור בטמפרטורת החדר. LiAc מגדיל את חדירות התא שמרים קיר ל-DNA. </li><li style=";text-align:right;direction:rtl"> פתרון 2: 0.1 M של ליתיום אצטט ו -50% פוליאתילן גליקול 4000. כמו כן פתרון 2 הוא פתרון עבודה הוא עשה ישירות האבקה. פתרון ללא מניות מוכנה. לשמור בטמפרטורת החדר. פיקדונות PEG oligos על תאים ושמרים. </li><li style=";text-align:right;direction:rtl"> RNA המכיל oligos (Thermo Scientific Dharmacon), 50-80, מרס, desalted, deprotected שאינם מטוהרים. Resuspend עד 250 pmoles / μl. חנות ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> ה-DNA רק oligos, 50-80, מרס, desalted שאינם מטוהרים. Resuspend עד 50 pmoles / μl. חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> SC-TRP (התקשורת להשלים סינתטי חסר טריפטופן) מדיה מוצק. </li><li style=";text-align:right;direction:rtl"> בקוטר 0.5 ס&quot;מ חרוזי זכוכית, מעוקרים ידי מעוקר. </li><li style=";text-align:right;direction:rtl"> RNase-off: פתרון RNase טיהור. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חופשי, מבחנות צנטריפוגה סטרילית. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חינם, צינורות חרוטי סטרילית. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חינם, פיפטה סטרילית אירוסול טיפים עם ZAP: 100-200 מ&quot;ל, 100-1000 מ&quot;ל. </li></ol> ב המושבה PCR חומרים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ה-DNA primers, desalted ולא מטוהרים. מתמוססים במים סטריליים עד 50 pmoles / μl. חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> תקי פולימראז ה-DNA, מאגר, dNTPs. </li><li style=";text-align:right;direction:rtl"> PCR צינורות. </li></ol> ג טיהור PCR. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> PCR ערכת טיהור. </li></ol> ד ג&#39;ל אלקטרופורזה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Agarose. </li><li style=";text-align:right;direction:rtl"> 1 x חיץ TBE ריצה (45 mM טריס-borate ו 1 ethylenediamine tetraacetate mM) מדוללת מ TBE 10x. </li><li style=";text-align:right;direction:rtl"> Prestained משקל מולקולרי הסמן. </li><li style=";text-align:right;direction:rtl"> ה-DNA לצבוע הטעינה. </li></ol> E. העיכול סייג. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגבלת אנזימים, מאגרים 10x, BSA. </li></ol> פ טיפול אלקליות עבור RNA המכיל אוליגו. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1 M NaOH של פתרון. </li><li style=";text-align:right;direction:rtl"> 1.2 מ &#39;של פתרון HCl. </li><li style=";text-align:right;direction:rtl"> 1 M של טריס-HCl, pH 7.4 פתרון. </li></ol>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

פולימרים סינתטיים קצר חומצות גרעין, oligonucleotides (oligos), הם כלי פונקציונלי ביותר נרחבת של הביולוגיה המולקולרית. Oligos יכול להיות מיוצר להכיל את כל ה-DNA הרצוי או רצף הרנ&quot;א יכול להיות מוכן לכלול מגוון רחב של הבסיס שינויים סוכר. יתר על כן, oligos ניתן שנועד לחקות שינויים ספציפיים חומצות גרעין, ולכן, יכול לשמש כלי חשוב לחקור את ההשפעות של נזק לדנ&quot;א מנגנוני התיקון. מצאנו כי Thermo Scientific Dharmacon RNA המכיל oligos עם אורך בין 50 ל 80 נוקלאוטידים יכול להיות מתאים במיוחד ללמוד, ב, פונקציות vivo וההשלכות של RNA כרומוזומליות / כלאיים דנ&quot;א של ribonucleotides מוטבע לתוך ה-DNA. RNA / DNA כלאיים יכול בקלות ליצור במהלך התיקון שכפול, שעתוק דנ&quot;א, לעומת זאת, מעט מאוד ידוע על היציבות של RNA / DNA כלאיים בתאים אשר במידה כלאיים אלה יכולים להשפיע על שלמות הגנטי של התאים. RNA המכיל oligos, אם כן, מייצגים וקטור המושלם להציג ribonucleotides לתוך הדנ&quot;א בכרומוזומים וליצור RNA / DNA כלאיים של אורך נבחר הרכב הבסיס. כאן אנו מציגים את הפרוטוקול עבור שילוב של ribonucleotides לתוך הגנום של שמרים מערכת אוקריוטים מודל / cerevisiae Saccharomyces /. עם זאת, במעבדה שלנו ניצלה Thermo Scientific Dharmacon RNA המכיל oligos לייצר RNA / DNA כלאיים ברמת כרומוזומליות במערכות תאים שונים, בין חיידקים לתאי האדם.

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

עבודה זו מראה כיצד ליצור כלאיים RNA / DNA ברמה כרומוזומליות ולחשוף העברת מידע גנטי מ-RNA ל-DNA הגנומי בתאי שמרים.

הדור של RNA / DNA כלאיים ב-DNA הגנום על ידי טרנספורמציה באמצעות RNA המכיל oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

generation-rnadna-hybrids-genomic-dna-transformation-using-rna

Research

JoVE Journal

Biology

26.8K Views.  Georgia Institute of Technology. עבודה זו מראה כיצד ליצור כלאיים RNA / DNA ברמה כרומוזומליות ולחשוף העברת מידע גנטי מ-RNA ל-DNA הגנומי בתאי שמרים.

Video: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Isolation of Genomic DNA from Mouse Tails

בידוד של הדנ&quot;א הגנומי של זנבות עכבר

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

טרנספורמציה של DNA פלסמיד לתוך החיידק שימוש בשיטה הלם חום

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

הפקת DNA גבוהה מולקולרית משקל הגנום מקרקעות ו משקעים

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

דור טראנסגנטי ג elegans על ידי שינוי Biolistic

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

הגנום MRI - משאב ציבורי עבור לימוד דפוסי בתוך רצף הדנ&quot;א הגנומי

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

בידוד ואפיון של RNA המכיל Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

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

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

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

שינוי גנטי של Picoeukaryote<em&gt; Ostreococcus tauri</em

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

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

לכידה סלקטיבית של 5-hydroxymethylcytosine מהדנ&quot;א הגנומי

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

הדמיה בזמן אמת של יחיד תכנון הנדסי RNA תמלילים בתאים חיים באמצעות משואות bimolecular רציומטרי

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

דור של מחיקות הגנומי בשורות תאים יונקים באמצעות CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...