Name: in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells
Uploaded: 2019-05-01T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

The project was funded by the National Institutes of Health (AI128406 www.nih.gov) to ZY and in part by Johnson Cancer Research Center (http://cancer.k-state.edu) in the form of Graduate Student Summer Stipend to PD.

1. Preparation of DNA Template by PCR for In Vitro Transcription
<ol>
	<li>To prepare the DNA template by PCR, design primers. When designing primers consider crucial characteristics like primer length, annealing temperature (Tm), GC content, 3&#8217; end with G or C etc. 
	NOTE: Discussed in detail in these literature25,26,27.</li>
	<li>Design primers to generate PCR amplicon ...

<ol>
	<li>Crick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+protein+synthesis.">On protein synthesis.</a> Symposia of the Society for Experimental Biology. 12, 138-163 (1958).</li><li>Crick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Dogma+of+Molecular+Biology.">Central Dogma of Molecular Biology.</a> Nature. 227, 561-563 (1970).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Translation+Initiation+in+Eukaryotes:+Mechanisms+and+Biological+Targets.">Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets.</a> Cell. 136, 731-745 (2009).</li><li>Spriggs, K. A., Bushell, M., Willis, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Regulation+of+Gene+Expression+during+Conditions+of+Cell+Stress.">Translational Regulation of Gene Expression during Conditions of Cell Stress.</a> Molecular Cell. 40, 228-237 (2010).</li><li>Shchelkunov, S. N., Marennikova, S. S., Moyer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Orthopoxviruses Pathogenic for Humans. , (2005).</li><li>Gale, M., Tan, S. L., Katze, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Control+of+Viral+Gene+Expression+in+Eukaryotes.">Translational Control of Viral Gene Expression in Eukaryotes.</a> Microbiology and Molecular Biology Reviews. 64, 239-280 (2000).</li><li>Walsh, D., Mathews, M. B., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+Translation:+Protein+Synthesis+in+Virus-Infected+Cells.">Tinkering with Translation: Protein Synthesis in Virus-Infected Cells.</a> Cold Spring Harbor Perspectives in Biology. 5, a012351 (2013).</li><li>Cao, S., Dhungel, P., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+against+the+Tide:+Selective+Cellular+Protein+Synthesis+during+Virally+Induced+Host+Shutoff.">Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.</a> Journal of Virology. 91, e00071-e00017 (2017).</li><li>Pelletier, J., Kaplan, G., Racaniello, V. R., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+translation+of+poliovirus+mRNA+is+conferred+by+sequence+elements+within+the+5&#8217;+noncoding+region.">Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5&#8217; noncoding region.</a> Molecular and Cellular Biology. 8, 1103-1112 (1988).</li><li>Pelletier, J., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+initiation+of+translation+of+eukaryotic+mRNA+directed+by+a+sequence+derived+from+poliovirus+RNA.">Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.</a> Nature. 334, 320-325 (1988).</li><li>Guo, L., Allen, E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+a+cap-independent+translation+element+that+functions+in+either+the+3&#8242;+or+the+5&#8242;+untranslated+region.">Structure and function of a cap-independent translation element that functions in either the 3&#8242; or the 5&#8242; untranslated region.</a> RNA. 6, 1808-1820 (2000).</li><li>Simon, A. E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3&#8242;+Cap-Independent+Translation+Enhancers+of+Plant+Viruses.">3&#8242; Cap-Independent Translation Enhancers of Plant Viruses.</a> Annual Review of Microbiology. 67, 21-42 (2013).</li><li>Marom, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+poly(A)+binding+proteins+mediate+internal+translational+initiation+by+a+plant+viral+IRES.">Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.</a> RNA Biology. 6, 446-454 (2009).</li><li>Liu, B., Qian, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+reprogramming+in+cellular+stress+response.">Translational reprogramming in cellular stress response.</a> Wiley Interdisciplinary Review. RNA. 5, 301-305 (2014).</li><li>Ahn, B. Y., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capped+poly(A)+leaders+of+variable+lengths+at+the+5&#8217;+ends+of+vaccinia+virus+late+mRNAs.">Capped poly(A) leaders of variable lengths at the 5&#8217; ends of vaccinia virus late mRNAs.</a> Journal of Virology. 63, 226-232 (1989).</li><li>Ahn, B. Y., Jones, E. V., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+vaccinia+virus+gene+encoding+an+18-kilodalton+subunit+of+RNA+polymerase+and+demonstration+of+a+5&#8217;+poly(A)+leader+on+its+early+transcript.">Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5&#8217; poly(A) leader on its early transcript.</a> Journal of Virology. 64, 3019-3024 (1990).</li><li>Schwer, B., Visca, P., Vos, J. C., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discontinuous+transcription+or+RNA+processing+of+vaccinia+virus+late+messengers+results+in+a+5&#8242;+poly(A)+leader.">Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5&#8242; poly(A) leader.</a> Cell. 50, 163-169 (1987).</li><li>Yang, Z., Martens, C. A., Bruno, D. P., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+initiation+and+3&#8242;+end+formation+of+poxvirus+post-replicative+RNAs.">Pervasive initiation and 3&#8242; end formation of poxvirus post-replicative RNAs.</a> Journal of Biological Chemistry. 287, 31050-31060 (2012).</li><li>Dhungel, P., Cao, S., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5&#8217;-poly(A)+leader+of+poxvirus+mRNA+confers+a+translational+advantage+that+can+be+achieved+in+cells+with+impaired+cap-dependent+translation.">The 5&#8217;-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.</a> PLOS Pathogens. 13, e1006602 (2017).</li><li>Jha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-kingdom+mimicry+underlies+ribosome+customization+by+a+poxvirus+kinase.">Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.</a> Nature. 546, 651-655 (2017).</li><li>Dai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+Profiling+Reveals+Translational+Upregulation+of+Cellular+Oxidative+Phosphorylation+mRNAs+during+Vaccinia+Virus-Induced+Host+Shutoff.">Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.</a> Journal of Virology. 91, e01858-e01816 (2017).</li><li>De Silva, F. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin-independent+plasmid+replication+occurs+in+vaccinia+virus+cytoplasmic+factories+and+requires+all+five+known+poxvirus+replication+factors.">Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors.</a> Journal of Virology. 2, 23 (2005).</li><li>Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-resolution+analysis+of+vaccinia+virus+and+host+cell+transcriptomes+by+deep+RNA+sequencing.">Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing.</a> Proceedings of the National Academy of Sciences. 107, 11513-11518 (2010).</li><li>Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Analysis+of+the+5&#8242;+and+3&#8242;+Ends+of+Vaccinia+Virus+Early+mRNAs+Delineates+Regulatory+Sequences+of+Annotated+and+Anomalous+Transcripts.">Genome-Wide Analysis of the 5&#8242; and 3&#8242; Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts.</a> Journal of Virology. 85, 5897-5909 (2011).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. , (2012).</li><li>Dieffenbach, C. W., Lowe, T. M., Dveksler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods and Applications. 3, S30-S37 (1993).</li><li>Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR Protocols: A Guide to Methods and Applications. , (2012).</li><li>Baklanov, M. M., Golikova, L. N., Malygin, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+on+DNA+Transcription+of+Nucleotide+Sequences+Upstream+to+T7+Promoter.">Effect on DNA Transcription of Nucleotide Sequences Upstream to T7 Promoter.</a> Nucleic Acids Research. 24, 3659-3660 (1996).</li><li>Thornton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+by+Overlap+Extension+PCR+to+Obtain+Hybrid+DNA+Products.">Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products.</a> The Genetic Manipulation of Staphylococci: Methods and Protocols. , 43-49 (2017).</li><li>Akichika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-specific+terminal+N6-methylation+of+RNA+by+an+RNA+polymerase+II&#8211;associated+methyltransferase.">Cap-specific terminal N6-methylation of RNA by an RNA polymerase II&#8211;associated methyltransferase.</a> Science. , (2018).</li><li>Sun, H., Zhang, M., Li, K., Bai, D., Yi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-specific,+terminal+N6-methylation+by+a+mammalian+m6Am+methyltransferase.">Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.</a> Cell Research. 1, (2018).</li><li>Boulias, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+m6Am+methyltransferase+PCIF1+reveals+the+location+and+functions+of+m6Am+in+the+transcriptome.">Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.</a> bioRxiv. , 485862 (2018).</li><li>Dominissini, D., Rechavi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N4-acetylation+of+Cytidine+in+mRNA+by+NAT10+Regulates+Stability+and+Translation.">N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation.</a> Cell. 175, 1725-1727 (2018).</li><li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+m6A,+m6Am,+and+m1A+Demethylation+Mediated+by+FTO+in+the+Cell+Nucleus+and+Cytoplasm.">Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.</a> Molecular Cell. 71, 973-985 (2018).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nature Reviews Genetics. 15, 293-306 (2014).</li></ol>

All four-core steps are critical to the success of the in vitro transcribed RNA-based luciferase reporter assay. Special attention should be given to primer design, especially for the T7 promoter sequence. T7 RNA polymerase starts transcription from the underlined first G (GGG-5&#39;-UTR-AUG-) in T7 promoter added before the 5&#39;-UTR sequence. Although the transcription start site (TSS) starts from the first G at the 5&#39; end, decreasing the number of G&#39;s less than three in T7 promoter region decreased the...

According to the central dogma, genetic information flows from DNA to RNA and then finally to protein1,2. This flow of genetic information is highly regulated at many levels including mRNA translation3,4. Development of reporter assays to measure regulation of gene expression will facilitate understanding of regulatory mechanisms involved in this process. Here we describe a protocol to study mRNA translation using an in vitro transcribed RNA-based luciferase reporter assay in poxvirus-infected cells.
Poxviruses comprise many highly dangerous human and animal pathogens5. Like all other viruses, poxviruses exclusively rely on host cell machinery for protein synthesis6,7,8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect it for translation of viral mRNAs7,8. One commonly employed mechanism by viruses is to use cis-acting elements in their transcripts. Notable examples include the Internal Ribosome Entry Site (IRES) and cap-independent translation enhancer (CITE)9,10,11. These cis-elements render the viral transcripts a translational advantage by attracting translational machinery via diverse mechanisms12,13,14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5&#8217;-untranslated region (5&#8217;-UTR): a 5&#8217;-poly(A) leader at the very 5&#8217; ends of these mRNAs15,16. The lengths of these 5&#8217;-poly(A) leaders are heterogeneous and are generated by slippage of the poxvirus-encoded RNA polymerase during transcription17,18. We, and others, recently discovered that the 5&#8217;-poly(A) leader confers a translation advantage to an mRNA in cells infected with vaccinia virus (VACV), the prototypic member of poxviruses19,20.
The in vitro transcribed RNA-based luciferase reporter assay was initially developed to understand the role of 5&#8217;-poly(A) leader in mRNA translation during poxvirus infection19,21. Although plasmid DNA-based luciferase reporter assays have been widely used, there are several drawbacks that will complicate the result interpretation in poxvirus-infected cells. First, plasmids are able to replicate in VACV-infected cells22. Second, cryptic transcription often occurs from plasmid DNA18,23,24. Third, VACV promoter-driven transcription generates poly(A)-leader of heterogeneous lengths consequently making it difficult to control the poly(A)-leader length in some experiments18. An in vitro transcribed RNA-based luciferase reporter assay circumvents these issues and the data interpretation is straightforward.
There are four key steps in this method: (1) polymerase chain reaction (PCR) to generate the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA; (3) transfection to deliver mRNA into cells; and (4) detection of luciferase activity as indicator of translation (Figure 1). The resulting PCR amplicon contains the following elements in 5&#8217; to 3&#8217; direction: T7-Promoter, poly(A) leader or desired 5&#8217;-UTR sequence, firefly luciferase open reading frame (ORF) followed by a poly(A) tail. PCR amplicon is used as the template to synthesize mRNA by in vitro transcription using T7 polymerase. During in vitro transcription, m7G cap or other cap analog is incorporated in newly synthesized mRNA. The capped transcripts are transfected into uninfected or VACV-infected cells. The cell lysate is collected at the desired time after transfection to measure luciferase activities that indicate protein production from transfected mRNA. This reporter assay can be used to study translation regulation by cis-element present in 5&#8217;-UTR, 3&#8217;-UTR or other regions of an mRNA. Furthermore, the in vitro transcribed RNA-based assay can be used to study different mechanisms of translation initiation including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation like IRES.

<table>
	<tbody>
		<tr>
			<td>2X-Q5 Master mix</td>
			<td>New England Biolabs</td>
			<td>M0492</td>
			<td>High-Fidelity DNA Polymerase used in PCR</td>
		</tr>
		<tr>
			<td>3&#180;-O-Me-m7G(5&#39;)ppp(5&#39;)G RNA Cap Structure Analog</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td>Anti reverse Cap analog or ARCA</td>
		</tr>
		<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>E.Z.N.A. Cycle Pure Kit</td>
			<td>OMEGA BIO-TEK</td>
			<td>D6492</td>
			<td>PCR purification kit</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>HiScribe T7 Quick High Yield RNA synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2050S</td>
			<td>In-Vitro transcription kit</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr>
			<td>NanoDrop2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td>Used to measure DNA and RNA concentration</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>Purelink RNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>12183018A</td>
			<td>RNA purification kit</td>
		</tr>
		<tr>
			<td>Vaccinia Capping System</td>
			<td>New England Biolabs</td>
			<td>M2080</td>
			<td>Capping system</td>
		</tr>
	</tbody>
</table>

in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

The four steps of in vitro transcribed RNA-based luciferase reporter assay: PCR to generate DNA template for In vitro transcription, in vitro transcription to generate mRNA, mRNA transfection, and luciferase measurement, can be seen in the schematic diagram (Figure 1). Designing of primers for both DNA templates (Fluc and Rluc) and the general scheme of overhang extension PCR is illustrated in the schematic (Figure 2A). After PCR...

Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

Our protocol for an in vitro transcribed RNA-based luciferase assay helps study translation regulation in pox virus infected cells. Additionally, a customized length of the Poly(A)liter allows studying its regulatory effects on translation. The main advantage of this technique is that it allows for the study of translation regulation by Cis elements such as 5'UTR and 3'UTR in mRNA and broad spectrum of translation initiation.The in vitro transcribed RNA-based luciferase reporter assay may be tailored to incorporate modification to cap and other modification and used to test the effect on translation. To begin, design forward and reverse primers to generate PCR amplicon containing the following elements in 5'to 3'direction:T7-Promoter, Poly(A)liter, Firefly Luciferase ORF in a Poly(A)tail referred to as T7_12A-Fluc, include several extra nucleotides in forward primer followed by T7-Promoter Poly(A)liter or desired 5'UTR sequence and approximately 20 nucleotides corresponding to the 5'end of the reporter gene's ORF. Adjust the primer length based on melting temperature corresponding to the 5'end of the reporter gene's ORF ensuring that the corresponding region in the primer is identical to the sense strand of the gene.Then design reverse primer to include Poly(A)tail and approximately 20 nucleotides. Adjust based on melting temperature corresponding to the 3'end of the reporter gene's ORF ensuring that the corresponding region of the primer is identical to the antisense strand of the gene and an in-frame stop codon is present before the Poly(A)tail. For internal control, design another set of primers containing the following elements in 5'to 3'direction:T7-Promoter, a random 5'UTR coding sequence containing Kozak sequence, Renilla Luciferase ORF, and Poly(A)tail, referred to as T7_Kozak-Rluc.To set up the reaction, add reagents in the following order to a PCR tube:DNase free water, 2X high-fidelity DNA polymerase, primers and sequence confirmed luciferase template DNA. After a standard three-step PCR cycle to generate a DNA template, detect the PCR product by running 5-10%of the PCR reaction in a 1%agarose TAE gel electrophoresis along with a molecular weight standard. To determine the size of the PCR product, visualize the gel under a UV illuminator.After determining the correct size of the PCR product, purify it using a commercially available PCR purification kit using 100 microliters of nuclease free water to elute the DNA. Check the concentration of the purified DNA using a spectrophotometer and store it at minus 20 degrees Celsius for use for in vitro transcription immediately. To synthesize RNA from the PCR product, add the following reagents to a microcentrifuge tube:DNase-RNase free water, NTP buffer mix, cap analog, PCR product template, and T7-RNA polymerase mix from in vitro transcription kit.Mix thoroughly. Incubate at 37 degrees Celsius for two hours and then proceed to the purification of the synthesized RNA using an RNA purification kit. To check the purified RNA, heat RNA at 70 degrees for five minutes to remove secondary structure and run it on 1.5 agarose TBE gel.Then visualize the gel under a UV illuminator. Check the concentration of the purified RNA using a spectrophotometer. Aliquot it and store at minus 80 degrees Celsius.Seed HeLa cells in a 24 well plate to achieve 80-90%confluency the following day and incubate overnight in an incubator at 37 degrees Celsius with 5%carbon dioxide. Infect HeLa cells with Vaccinia virus at a multiplicity of infection of five and keep uninfected HeLa cells for comparison. Then incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 10 to 12 hours.After desired hours post infection, mix 480 nanograms of 12A sequence bearing Firefly Luciferase mRNA and 20 nanograms of Kozak sequence bearing Renilla Luciferase mRNA in one microcentrifuge tube. In another microcentrifuge tube, add 1.1 microliters of cationic lipid transfection reagent. Add 55 microliters of reduced serum media to both tubes, mix and incubate at room temperature for five minutes.Then add 55 microliters cationic lipid transfection reagent containing reduced serum media to mRNA containing tube. Mix gently but thoroughly with a pipette and incubate at room temperature for 15 minutes. During the incubation, remove the cell culture medium from the cells and add 400 microliters of reduced serum medium per well.When incubation is completed, add 100 microliters of the mixture drop wise and evenly per well of the 24 well plate. Five hours post co-transfection with 12A Fluc and Kozak Rluc mRNA, remove the reduced serum medium from the cells and lyse the cells by adding 150 microliters 1X lysis buffer from the Luciferase assay kit capable of performing two reporter assays. After incubating for 10 minutes at room temperature, scrape the cells using rubber and sterile syringe and transfer to a microcentrifuge tube.To pellet cell debris, centrifuge the lysate at 12, 000 times g for 10 minutes at four degrees Celsius. Transfer 30 microliters of supernatant per well of an opaque walled 96 well white assay plate with a solid bottom. Use the Luciferase assay kit and a multi-mode plate reader luminometer to measure the dual luminescence using kinetics function as described in the manuscript.After exporting the luminescence reading data into a desirable file format, determine relative translation rate from 12A Fluc mRNA in uninfected and VACV infected HeLA cells by dividing Fluc value by internal control Rluc value. This assay was used to test the translation efficiency of an Fluc mRNA that contains a 5'Poly(A)liter in uninfected and VACV infected cells. Both uninfected and VACV infected cells were successfully co-transfected with Fluc and Rluc mRNA.Dividing Fluc by Rluc normalized the transfection efficiency in RNA stability in cells. 5'Poly(A)liter containing mRNA has a translational advantage during VACV infection compared to uninfected cells. This was not due to differential transfection efficiency or mRNA stability as the RNA level was similar in uninfected and VACV infected cells five hours post mRNA transfection.Take special care when designing primers as this can affect not only PCR reaction efficiency but also all downstream processes requiring the amplified DNA. This method is an important assay to quickly test the translation regulation by Cis elements. If possible, this method should be corroborated by other complementary experiments.Precaution should be taken while using Vaccinia virus and avoid exposure to chemicals such as ethidium bromide and phenol.

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Immunology and Infection

Unit 1

Translation: RNA to Protein

SCIENTISTS IN ACTION

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...