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EoE Sub Articles

1. Transfect mRNA to Cells
<ol>
	<li>Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison. 
	NOTE: MOI is the number of infectious viral particles per cell.</li>
	<li>MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} &#181;l of virus per 1 mL medium.</li>
	<li>After desired h post-infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well of 24-well plates) using a cationic lipid transfection reagent as shown in Figure 1C.
	<ol>
		<li>For one well of a 24-well plate, mix 480 ng of 12A sequence bearing firefly luciferase (12A-Fluc) mRNA and 20 ng of Kozak sequence bearing Renilla luciferase (Kozak-Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 &#181;L of cationic lipid transfection reagent.</li>
		<li>Add 55 &#181;L of reduced serum medium in both tubes. Mix and incubate at room temperature for 5 min.</li>
		<li>After 5 min of incubation, add 55 &#181;L cationic lipid transfection reagent containing reduced serum medium in mRNA mRNA-containing tube.</li>
		<li>Mix gently but thoroughly, and incubate at room temperature for 15 min.</li>
		<li>During the incubation, remove the cell culture medium and add 400 &#181;L of reduced serum medium per well of 24-well plates.</li>
		<li>After incubation, add 100 &#181;L of the mixture dropwise and evenly to one well of 24-well plates.</li>
	</ol>
	</li>
</ol>
2. Measure Luciferase Activities
<ol>
	<li>Five hours post-co-transfection of 12A-Fluc and Kozak-Rluc mRNA, measure luciferase activity using a luciferase assay system capable of performing two reporter assays (e.g., Dual-Luciferase Reporter assay kit).</li>
	<li>Remove the reduced serum medium and lyse the cells by adding 150 &#181;L 1x lysis buffer, a component of the luciferase assay kit.</li>
	<li>After 10 min incubation at room temperature, collect the lysate by scrapping the cells and transfer them to a microcentrifuge tube.</li>
	<li>Centrifuge the lysate at 12,000 x g for 10 min at 4 &#176;C to pellet cell debris.</li>
	<li>Add 30 &#181;L of supernatant in an opaque-walled 96-well white assay plate with a solid bottom.</li>
	<li>Measure the dual luminescence using the luciferase assay kit and a multimode plate reader luminometer.</li>
	<li>Perform the measurement using the kinetics function (on a per-well basis) using the settings described in Table 1. 
	NOTE: The reading can also be taken using a manual luminometer. Add an equal volume of lysate and substrate for Fluc in a cuvette. Wait for 2 s and measure for 10 s using a luminometer. Following the Fluc measurement, quickly take out the cuvette from the luminometer and add an equal volume of the substrate for Rluc manually. Again, wait for 2 s and measure for 10 seconds using a luminometer.</li>
	<li>Export the luminescence reading data into a desirable file format.</li>
	<li>Determine the relative translation rate from 12A-Fluc mRNA in uninfected and VACV-infected HeLa cells by dividing the Fluc value by the internal control Rluc value. 
	NOTE: Figure 2 shows the step-by-step analysis of raw data to get relative Fluc activity.</li>
</ol>
Table 1: Luciferase Measurement Settings &#8211; the steps for luciferase measurement with the recommended volume or time.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Steps</td>
			<td>Volume/Time</td>
		</tr>
		<tr>
			<td>Inject Luciferase Assay Substrate (Fluc):</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>Wait / Incubation time:</td>
			<td>2 s</td>
		</tr>
		<tr>
			<td>Luminescence Measurement (Fluc):</td>
			<td>10 s</td>
		</tr>
		<tr>
			<td>Stop &#38; Glo Substrate (Rluc):</td>
			<td>30 &#181;L</td>
		</tr>
		<tr>
			<td>Wait / Incubation time:</td>
			<td>2 s</td>
		</tr>
		<tr>
			<td>Luminescence Measurement (Rluc):</td>
			<td>10 s</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning 96 Well Half-Area white flat bottom polystyrene microplate</td>
			<td>Corning</td>
			<td>3693</td>
			<td>Opaque walled 96 well white plate with solid bottom</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>Dual-Luciferase Assay Kit (DLAK)</td>
		</tr>
		<tr>
			<td>GloMax Navigator Microplate Luminometer</td>
			<td>Promega</td>
			<td>GM2010</td>
			<td>Referred as multimode plate reader luminometer</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Reduced serum media</td>
		</tr>
	</tbody>
</table>

a luciferase reporter assay to study translation regulation in poxvirus-infected cells

Source: Dhungel, P., et al. <a href="https://app.jove.com/v/59626">In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells.</a> J. Vis. Exp. (2019)
This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5&#39; Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader sequence.

<img alt="Figure 1" src="/files/ftp_upload/22002/22002_Figure1.jpg" /> 
Figure 1: mRNA synthesis and transfection. (A) Schematic of in vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5&#39;-UTR of interest and the T7 promoter is used as the template. The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5&#39; to 3&#39; direction. Once mRNA is 25-30 nt l...

A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

1. Transfect mRNA to Cells

	Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at ...

Take transfection complexes comprising reporter mRNAs encoding for firefly luciferase, or Fluc, and Renilla luciferase, or Rluc.
Fluc mRNA contains a 5&#39; polyadenosine sequence, or a poly(A) leader, while Rluc mRNA lacks this sequence.
Transfect uninfected and poxvirus-infected mammalian cells to deliver the reporter mRNAs, and incubate.
In uninfected cells, eukaryotic initiation factors bind to the mRNA&#39;s 5&#39; cap, followed by recruitment of the small ribosomal subunit and an initiator tRNA.
Upon the small subunit locating the start codon, the large subunit joins to synthesize luciferase proteins.
In infected cells, poxvirus-mediated phosphorylation of the small subunit causes ribosome assembly directly on the leader sequence, enhancing Fluc production.
Lyse the cells to release the luciferases.
Add the Fluc substrate, which the enzyme oxidizes, emitting light. Using a plate reader, measure the luminescence.
Repeat the step for Rluc to determine the relative increase in the Fluc luminescence compared to Rluc in virus-infected cells.

a-luciferase-reporter-assay-to-study-translation-regulation-poxvirus

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

148 Aufrufe. Quelle: Dhungel, P., et al. In-vitro transkribierter RNA-basierter Luciferase-Reporter-Assay zur Untersuchung der Translationsregulation in Pockenvirus-infizierten Zellen. J. Vis. Exp. (2019) Dieses Video zeigt einen Assay zur Untersuchung der Translationsregulation in Pockenvirus-infizierten Zellen. Nicht infizierte und virusinfizierte Zellen wurden mit mRNAs co-transfiziert, die für Glühwürmchen-Luziferase (Fluc) und Renilla-Luziferase (Rluc) ko-transfizieren, wobei die Fluc-mRNA eine 5'-P...

Serie: Ein Luciferase-Reporter-Assay zur Untersuchung der Translationsregulation in Pockenvirus-infizierten Zellen - Konzept

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Quantitative Immunfluoreszenz Maßnahme Global lokalisierte Übersetzung

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Forschung

JoVE Journal

Biochemie

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Toeprinting Analyse der Übersetzung Einleitung Komplexbildung auf Säugetier-mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

Transkript

A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells