This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

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The authors have nothing to disclose.

פרוטוקול זה מדגיש במבחנה ובשירות vivo של GLuc-SERCaMP לפקח דלדול סידן ER. למרות שינוי החלבון כדי ליצור SERCaMP מופיע להכליל לחלבוני כתב אחרים 12, בחרנו בלוציפראז Gaussia לפליטת האור שלו חזק (200-1,000 הגדול יותר פי) בהשוואה לluciferases האחר 18. אנו מדגימים שחרור GLuc-SERCaMP זיה?...

Reticulum endoplasmic (ER) פונקציות רבות יכולות סלולריות כוללים קיפול חלבונים, הפרשת חלבון, שומנים הומאוסטזיס, ואיתות תאית 1. מרכז לתפקוד נורמלי ER הוא שמירת ריכוזי סידן luminal ב ~ 5000 פעמים אלה שנמצאו בציטופלסמה 2-4. תהליך עתיר אנרגיה זה מוסדר על ידי ATPase סידן Sarco / reticulum endoplasmic (SERCA), משאבה שזז יוני סידן לחדר המיון. זרימה של סידן מחדר המיון מתווכת בעיקר על ידי ryanodine (RyR) וקולטני אדנוזין אינוזיטול (IP3R). בגלל תהליכי מיון רבים תלויים בסידן, לשבש את החנות יכולה להוביל ללחץ ER ומוות של תאי סופו של דבר. ER חוסר ויסות הסידן נצפה במחלות כולל קרדיומיופתיה, סוכרת, אלצהיימר, פרקינסון ושל 5. בשל הפרוגרסיביות של מחלות אלו, זה כבר מאתגר להתוות סיבה והתוצאה מחדשlationship בין פתוגנזה ושינויים בחנות סידן ER. מספר הטכנולוגיות איפשר להתקדמות משמעותית בהבנה של דינמיקת סידן ER, כוללים צבעים ואינדיקטורים מקודדים גנטיים סידן (GECIs) שלנו. צבעים נמוכים זיקת סידן, המגדילים בקרינה כאשר חייב Ca 2 +, ניתן לטעון לתאים לבחון תאי subcellular עם ריכוזים גבוהים של סידן 6. GECIs, כגון D1ER ותופס לאפשר ניטור של תנודות סידן עם שליטה מדויקת יותר של לוקליזציה subcellular 7-9. לאחרונה, סוג אחר של GECIs נקראים אינדיקטורים למדידת סידן בפח אברון חלבון (CEPIA) תוארו 10. גישה שלישית המשלבת גנטיקה וכימיה מולקולה קטנה הוא טעינת צבע הממוקד-אסטראז (TED), אשר מנצל carboxylesterase מקודדים גנטי (ממוקד לחדר המיון) עם צבע סידן מבוסס אסתר 11. בעוד המגייסיש גישות ementioned נקודות חוזק וחולשה טבועים, הם יכולים לספק תובנה חשובה דינמיקת סידן ER באמצעות מדידות של הקרינה אקוטיות. הם, לעומת זאת, לא אופטימליים למחקרים ארוכי טווח לעתים קרובות נדרשו לחקור את התקדמות מחלה. במטרה להמציא שיטה לעקוב אחר דינמיקת סידן על פני תקופות זמן ארוכות, זיהינו ופיתחנו שינוי חלבון ליצירת חלבוני סידן מופרש ER ניטור (SERCaMPs) 12. SERCaMP עוקף כמה מגבלות הקשורות לשיטות אחרות, על ידי מתן גישה פולשנית לחקור חנות סידן ER שוב ושוב. אנחנו הוכחנו בעבר כי ASARTDL carboxy מסוף פפטיד (אלאנין-סרין-אלאנין-ארגינין-תראונין-אספרטית חומצה-לאוצין) מספיק כדי לקדם את שימור ER; עם זאת, בתנאים שגורמים לירידות בסידן ER, רצף הפפטיד הוא כבר לא מסוגל לשמור על localizatio ERn והחלבון מופרש 13. בסיס טכנולוגית SERCaMP הוא התוספת של ASARTDL לcarboxy-הסופית של חלבון מופרש (לוציפראז למשל Gaussia, או GLuc) כך שההפרשה מופעלת על ידי דלדול סידן ER, ובכך ליצור כתב חזק של חוסר ויסות הסידן ER 12. הביטוי של GLuc-SERCaMP באמצעות שיטות מהונדסים מאפשר נוזלים ביולוגיים כוללים תרבית תאים בינוני ופלזמה להיות מנותח לשינויים בפעילות GLuc כאינדיקטור של הומאוסטזיס סידן ER. יש שיטת בקשות למחקר האורך של שינויים הדרגתיים בחנות סידן ER הן במבחנה in vivo. הפרוטוקול הבא נכתב כמתווה כללית לשימוש SERCaMP מבוסס GLuc ללמוד הומאוסטזיס סידן ER, אבל הפרוטוקול יכול לשמש כמדריך לSERCaMPs כתב חלופי.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.5mL tubes</td>
			<td>Fisher&#160;</td>
			<td>02-682-550</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40 solution&#160;</td>
			<td>Pierce</td>
			<td>28324</td>
			<td>for intracellular GLuc assays</td>
		</tr>
		<tr>
			<td>1mL luer-lok syringes</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL filter tips</td>
			<td>Rainin</td>
			<td>RT-L200F</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 surgical sutures</td>
			<td>Fisher</td>
			<td>NC9598192</td>
			<td></td>
		</tr>
		<tr>
			<td>30g needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microplate sealing sheets</td>
			<td>Thermo</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>Fisher</td>
			<td>22-246-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Auto Flow System</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-AF9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal recovery chamber</td>
			<td>Lyon Vet</td>
			<td>ICU-912-004</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher</td>
			<td>NC9386574</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine growth serum</td>
			<td>Thermo</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine, Native</td>
			<td>Regis Technologies</td>
			<td>1-361204-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting needles 3/8 circle sutures</td>
			<td>WPI</td>
			<td>501803</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital ultrasconic cleaner</td>
			<td>Fisher Scientific</td>
			<td>FS60D</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose, GlutaMAX, pyruvate</td>
			<td>Life Technologies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA mass ladder</td>
			<td>Life Technologies</td>
			<td>10496-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase (recombinant protein)</td>
			<td>Nanolight</td>
			<td>321-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td>
			<td>New England Biolabs</td>
			<td>E8023S</td>
			<td>1:2000 (WB)</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>CellPoint Scientific</td>
			<td>DS-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluc assay plates (96 well, opaque)</td>
			<td>Fisher</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Allmedtech</td>
			<td>63323-276-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butler Schein</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>995-2949</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik Stop Styptic powder</td>
			<td>Butler Schein</td>
			<td>5867</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>a452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 22R Centrifuge</td>
			<td>Bekman Colter</td>
			<td>368831</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Fisher</td>
			<td>19-898-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Gel and PCR Cleanup</td>
			<td>Machery-Nagel</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet</td>
			<td>Rainin</td>
			<td>L-200XLS+</td>
			<td></td>
		</tr>
		<tr>
			<td>p24 Lenti-X rapid titer kit</td>
			<td>Clontech</td>
			<td>632200</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR film seal</td>
			<td>Fisher</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>ReFresh Charcoal Filter canister</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades, #10</td>
			<td>Fine Science tools Inc</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats 150-200g</td>
			<td>Charles River</td>
			<td>Rats</td>
			<td>rats ordered at 150-200g.&#160; Surgery 5 days after arrival</td>
		</tr>
		<tr>
			<td>Small animal ear tags</td>
			<td>National Band and Tag co</td>
			<td>1005-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drapes</td>
			<td>Braintree Scientific</td>
			<td>SP-MPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 2 plate reader</td>
			<td>BioTek</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma</td>
			<td>T9033</td>
			<td>harmful to human health</td>
		</tr>
		<tr>
			<td>Virapower lentiviral packaging mix</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xfect Transfection reagent</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Valley Vet</td>
			<td>468RX</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp

Reticulum endoplasmic (ER) מכיל את הרמה הגבוהה ביותר של סידן תוך תאי, עם ריכוזים כ -5,000 פי יותר מרמות cytoplasmic. פיקוח הדוק על סידן ER הכרחי לקיפול חלבונים, שינוי וסחר. הפרעות לסידן ER יכולות לגרום להפעלה של תגובת החלבון מקופלת, מנגנון תגובת לחץ ER שלוש שיניים, ותורמות לפתוגנזה במגוון רחב של מחלות. היכולת לעקוב אחר שינויי סידן ER במהלך התפרצות מחלה והתקדמות חשובה בעיקרון, אך מאתגרת בפועל. שיטות זמינות כעת עבור סידן ER הניטור, כגון צבעי ניאון סידן תלוי וחלבונים, סיפקו תובנה דינמיקת סידן ER בתאים, אולם הכלים אלה אינם מתאימים ללימודים בvivo. המעבדה שלנו הוכיחה ששינוי לcarboxy-הסופית של לוציפראז Gaussia מקנה הפרשת הכתב בתגובה לדלדול סידן ER. השיטות לשימוש בלוציפראז מבוסס, חלבון ניטור סידן מופרש ER (SERCaMP) במבחנה וביישומי vivo מתוארות במסמך זה. וידאו זה מדגיש זריקות כבד, מניפולציה תרופתית של GLuc-SERCaMP, איסוף דם ועיבוד, ופרמטרי assay לניטור אורך של סידן ER.

שיטת GLuc-SERCaMP מאפשרת הערכה של הומאוסטזיס סידן ER על ידי דגימת נוזלים תאיים. ניתן לכלול מספר פקדים בעיצוב ניסיוני כדי לשפר את הפרשנות של תוצאות. ראשית, שימוש בכתב constitutively מופרש (למשל GLuc ללא ASARTDL C-המסוף או &quot;GLuc-אין תג&quot;) יכול להיות מועסק על מנת להעריך את ההשפעות של טי...

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

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 ...

monitoring-endoplasmic-reticulum-calcium-homeostasis-using-gaussia

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Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

13.1K Views.  National Institutes of Health. 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.

Video: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

לימוד biogenesis ממברנה עם Assay בלוציפראז מבוסס Gene כתב

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

ניטור שינויים דינמיים רמות הסידן מיטוכונדריאלי במהלך אפופטוזיס באמצעות חיישן סידן מקודד גנטית

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

בתעתיק במבחנה תקרת של mRNA בלוציפראז Gaussia ואחריו transfection Cell הלה

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

ויזואליזציה של mRNAs לוקליזציה reticulum endoplasmic בתאי יונקים

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

הקרנה פונקציונלית תפוקה גבוהה באמצעות assay בלוציפראז Dual-זוהר תוצרת בית

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

ויזואליזציה של תחומי משנה reticulum endoplasmic בתאים בתרבית

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfecting תאי RAW264.7 עם לוציפראז כתב ג&#39;ין

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

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, ...

ניטור הומאוסטזה סידן endoplasmic reticulum שימוש

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Chapters in this video

לימוד biogenesis ממברנה עם Assay בלוציפראז מבוסס Gene כתב

ניטור שינויים דינמיים רמות הסידן מיטוכונדריאלי במהלך אפופטוזיס באמצעות חיישן סידן מקודד גנטית

בתעתיק במבחנה תקרת של mRNA בלוציפראז Gaussia ואחריו transfection Cell הלה

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

ויזואליזציה של mRNAs לוקליזציה reticulum endoplasmic בתאי יונקים

הקרנה פונקציונלית תפוקה גבוהה באמצעות assay בלוציפראז Dual-זוהר תוצרת בית

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

ויזואליזציה של תחומי משנה reticulum endoplasmic בתאים בתרבית

Transfecting תאי RAW264.7 עם לוציפראז כתב ג'ין

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