עבודה זו נתמכה בחלקו על ידי הקרן הלאומי למדע (IOS-0920417) (ACL).

אין ניגודי האינטרסים הכריזו.

1. עשיית שינויים בפרוטוקול הנוכחי 1.1 מכשירי הקלטה ושיקולי תפוקה בגלל הזמינות המסחרית שלה, LumiCycle (Actimetrics) הפך את מכשיר luminometer האוטומטי הנפוץ ביותר להקלטה בזמן אמת 4,5,9,19,29-31. LumiCycle מעסיק צינורות ...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> DMEM </td><td> HyClone </td><td> SH30243FS </td><td> לצמיחת תאים רגילות </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100-046 </td><td> לluminometry </td></tr><tr><td> FBS </td><td> HyClone </td><td> SH3091003 </td><td></td></tr><tr><td> עט / דלקת / Gln (100x) </td><td> HyClone </td><td> SV3008201 </td><td></td></tr><tr><td> B-27 </td><td> Invitrogen </td><td> 17504-044 </td><td></td></tr><tr><td> D-luciferin </td><td> ביוסינט </td><td> L-8220 </td><td></td></tr><tr><td> פולי-L-ליזין </td><td> סיגמא </td><td> P4707 </td><td></td></tr><tr><td> Polybrene </td><td> Millipore </td><td&gt; TR-1003-G </td><td></td></tr><tr><td> Forskolin </td><td> סיגמא </td><td> F6886 </td><td></td></tr><tr><td> כל כימיקלים אחרים </td><td> סיגמא </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> ציוד </td></tr><tr><td> רקמת תרבות חממה </td><td></td><td></td><td> 5% CO2 על 37 מעלות צלזיוס </td></tr><tr><td> ברדס תרביות רקמה </td><td></td><td></td><td> מנהלת הליגה מוסמכת-2 </td></tr><tr><td> מיקרוסקופ אור וניאון </td><td></td><td></td><td> לעומת שלב אופציונלי </td></tr><tr><td> LumiCycle </td><td> Actimetrics </td><td></td><td></td></tr></table>

monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters

ביונקים, היבטים רבים של התנהגות ופיזיולוגיה כגון מחזורי שינה והערות וחילוף חומרים כבדים מוסדרים בשעוני יממת אנדוגני (סקר 1,2). המערכת הביולוגית זמן שמירה, היא רשת היררכית רב מתנד, עם השעון המרכזי ממוקם בגרעין suprachiasmatic (SCN) סנכרון ותיאום שעונים-SCN נוסף והיקפיים אחר 1,2. תאים בודדים הם יחידות פונקציונליות לדור ותחזוקה של מקצבי יממה 3,4 ומתנדים אלה של סוגי רקמות שונים בנתח אורגניזם מנגנון משוב דומה להפליא ביוכימי שלילי. עם זאת, בשל אינטראקציות ברמת הרשת העצבית בSCN ובאמצעות רמזים קצביים, מערכתיים ברמת האורגניזם, מקצבי יממה ברמת האורגניזם אינם בהכרח תא אוטונומי 5-7. בהשוואה למחקרים מסורתיים של פעילות של תנועה בגוף חי וexplants SCN vivo לשעבר, גell מבוסס במבחני חוץ גופייה מאפשרת גילוי פגמי תא אוטונומי יממת 5,8. מבחינה אסטרטגית, מודלים מבוססי תאים יותר בניסוי צייתנים לאפיון פנוטיפי וגילוי מהיר של מנגנוני שעון בסיסיים 5,8-13. בגלל מקצבי יממה הם דינמיים, מדידות אורכות עם רזולוציה גבוהה זמנית נחוצות להערכת תפקוד שעון. בשנים האחרונות, הקלטת פליטת אור בזמן אמת באמצעות בלוציפראז הגחלילית ככתב הפכה לשיטה נפוצה ללימוד מקצבי יממה ביונקים 14,15, שכן היא מאפשרת לבחינה של ההתמדה והדינמיקה של מקצבים מולקולריים. כדי לפקח על מקצבי יממת תא אוטונומי של ביטוי גנים, כתבי וציפראז ניתן הוכנסו לתאים באמצעות transfection החולף 13,16,17 או תמרה יציבה 5,10,18,19. כאן אנו מתארים פרוטוקול תמרה יציבה באמצעות משלוח הגן lentivirus בתיווך. Tמערכת וקטור lentiviral הוא עדיף על שיטות מסורתיות כגון transfection חולף ושידור germline בגלל היעילות והרבגוניות שלו: הוא מאפשר העברה יעילה ויציבה אינטגרציה לתוך הגנום של המארח הוא חלוקה ולא חלוקת תאים-20. ברגע ששורת תאי כתב הוא הקים, את הדינמיקה של פונקציית שעון יכולה להיבחן דרך הקלטת פליטת אור. אנו מתארים לראשונה את הדור (Per2) קווי P-d לוק כתב, ולאחר מכן מציגים נתונים מזה וכתבי יממה אחרים. במבחנים אלה, fibroblasts העכבר 3T3 וU2OS תאי סרטן עצמות אדם משמשים כמודל לסלולרי. אנחנו גם לדון בדרכים שונות של שימוש במודלים אלה במחקרי שעון יממה. שיטות שתוארו כאן ניתן ליישם במגוון רחב של סוגי תאים ללמוד את הבסיס התאי ומולקולרי של שעוני יממה, ועשויה להיות שימושי בהתמודדות עם בעיות במערכות ביולוגיות אחרות.

Watch this Scientific Journal Video about Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters at JoVE.com

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

שעוני יממה לתפקד בתוך תאים בודדים, כלומר, הם תאים אוטונומיים. כאן, אנו מתארים שיטות ליצירת מודלי שעון תא אוטונומי באמצעות טכנולוגיה לא פולשנית, לוציפראז מבוסס זמן אמת פליטת אור. תאי Reporter מספקים מערכות צייתניות, פונקציונליות מודל ללימוד ביולוגית יממה.

ניטור מקצבי Cell-אוטונומי יממת שעון של ביטוי גנים באמצעות כתבי פליטת האור לוציפראז

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

monitoring-cell-autonomous-circadian-clock-rhythms-gene-expression

Research

JoVE Journal

Biology

22.1K Views.  The University of Memphis. שעוני יממה לתפקד בתוך תאים בודדים, כלומר, הם תאים אוטונומיים. כאן, אנו מתארים שיטות ליצירת מודלי שעון תא אוטונומי באמצעות טכנולוגיה לא פולשנית, לוציפראז מבוסס זמן אמת פליטת אור. תאי Reporter מספקים מערכות צייתניות, פונקציונליות מודל ללימוד ביולוגית יממה.

Video: Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Bacterial Gene Expression Analysis Using Microarrays

Gene Expression חיידקית ניתוח שימוש microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

מדידה של קצב חילוף החומרים ב<em&gt; דרוזופילה</em&gt; באמצעות Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

ניטור הומאוסטזה סידן endoplasmic reticulum שימוש<em&gt; Gaussia</em&gt; לוציפראז SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

זיהוי של ביטוי גנים בתאים תאיים חיים של Murine, אדם וחזירי מקור באמצעות חלקיקים שכותרתו פלואורסצנטי

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

ניתוח מהיר של פנוטיפים היממה ארבידופסיס פרוטופלאסט טרנספקציה עם כתבת שעון פלורסנט

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...