Name: live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2
Uploaded: 2021-11-05T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

We would like to thank members at our institute (Texas Biomedical Research Institute) for their efforts in keeping our facilities fully operational and safe during the COVID-19 pandemic. We would also like to thank our Institutional Biosafety Committee (IBC) and spell (IACUC) for reviewing our protocols in a time-efficient manner.We thank Dr. Thomas Moran at the Icahn School of Medicine at Mount Sinai for providing the SARS-CoV cross-reactive 1C7C7 nucleocapsid (N) protein monoclonal antibody. SARS-CoV-2 research in the Martinez-Sobrido&#39;s laboratory is currently supported by the NIAID/NIH grants RO1AI161363-01, RO1AI161175-01A1, and R43AI165089-01; the Department of Defense (DoD) grants W81XWH2110095 and W81XWH2110103; the San Antonio Partnership for Precision Therapeutic; the Texas Biomedical Research Institute Forum; the University of Texas Health Science Center at San Antonio; the San Antonio Medical Foundation; and by the Center for Research on Influenza Pathogenesis and Transmission (CRIPT), a NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract # 75N93021C00014).

Protocols involving K18 hACE2 transgenic mice were approved by the Texas Biomedical Research Institute (TBRI) Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC). All experiments follow the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council40. The appropriate Personal Protection Equipment (PPE) is required when working with mice.
1. Use of K18 hACE2 transgenic mice
<ol>
	<li>Purchase and maintain 4-6-week-old female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18 hACE2 transgenic mice) in a biosafety level (BSL)-2 animal care facility under specific pathogen-free conditions.
	<ol>
		<li>After arrival at the BSL2 facilities, allow the animals to acclimate for 7 days and transfer them to the BSL-3 animal facility for infections and other experimental procedures.</li>
		<li>Following IACUC protocols, place 4 mice per cage. To ensure that the animal is deceased, after mice infection with rSARS-CoV-2 and in vivo imaging, euthanize the animals with a lethal dose of Fatal-Plus (&#62;100 mg/kg).</li>
	</ol>
	</li>
</ol>
2. Biosafety
NOTE: In this manuscript, rSARS-CoV-2 is generated using the BAC-based reverse genetic systems for SARS-CoV-2 USA-WA1/2020 strain, as previously described37. All in vivo procedures involving rSARS-CoV-2/Nluc or rSARS-CoV-2/Venus infections must be performed in a biological safety cabinet under BSL-3 conditions.
<ol>
	<li>Clean the biosafety cabinet with 2% Wexicide and 70% Ethanol disinfectant sequentially before and after performing all the experimental procedures described in this article.</li>
	<li>Sterilize all dissection material (scissors, dissecting forceps, etc.) and the homogenizer before and after each use.</li>
	<li>Clean the isolation chamber and disinfect using MB10 tablets before and after each use according to the manufacturer&#39;s instruction.</li>
	<li>Discard all biological material produced during the procedures following IBC and IACUC guidelines.</li>
</ol>
3. In vivo characterization of rSARS-CoV-2
<ol>
	<li>Mouse infections
	<ol>
		<li>Place female 4-6-week-old K18 hACE2 transgenic mice in labeled cages, identify the mice in each cage using an ear punch code and place 4 mice per cage. Use a group of mice for body weight and survival and another group of animals for viral titration and imaging.</li>
		<li>Before infection, shave mice chest on the ventral side from the pectoral nipple to the inguinal nipple area to facilitate the bioluminescence signal.</li>
		<li>Prepare the rSARS-CoV-2 inoculum with 105 plaque-forming units (PFU)/mouse under sterile conditions in a total volume of 50 &#181;L using sterile 1x phosphate buffer saline (PBS). 
		NOTE: Calculate the amount of rSARS-CoV-2 to be used in the viral dilution with the formula: virus needed = (105 PFU per mouse / 50 &#181;L) x final volume) / stock viral titer.</li>
		<li>Keep the virus inoculum chilled on ice.</li>
		<li>Use the mock-infected (1x PBS) and rSARS-CoV-2/WT-infected mice as internal controls. Place the mock-infected mice in a separate cage than rSARS-CoV-2-infected mice.</li>
		<li>Anesthetize the mice using 5% isoflurane gaseous sedation in an anesthesia chamber and maintain with 3% isoflurane.</li>
		<li>Once postural reaction and righting reflex are confirmed, place the mouse in the dorsal recumbency position.</li>
		<li>Scruff the neck of the mouse between the index finger and thumb and hold the tail against the palm of the hand with the pinky finger to hold the animal in a dorsal position.</li>
		<li>Place the pipette tip containing 50 &#181;L of the virus inoculum in the nostril and slowly eject the solution. Ensure that the virus inoculum is inhaled and there is no reflux by observing the inoculum drop disappearing.</li>
	</ol>
	</li>
	<li>Bioluminescence monitoring K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 1 and Figure 2) 
	NOTE: This experiment follows the schematic representation and uses the viruses displayed in Figure 1. An isoflurane anesthesia attachment is required for the in vivo imaging system (IVIS). See Table of Materials for details instruments and systems required.
	<ol>
		<li>Initiate the imaging software and set up the parameters, click image mode to Bioluminescence, open filter, and set the exposure time to auto.</li>
		<li>Upon initializing the IVIS machine, place the mice in the isolation chamber while still inside the biosafety cabinet. Induce mice with isoflurane at a 5% concentration. Once loss of the postural reaction and righting reflex is confirmed, maintain with 3% isoflurane.</li>
		<li>Once mice are anesthetized, remove them from the isolation chamber and retro-orbitally administer the luciferase substrate diluted 1:10 in 1x PBS (final volume 100 &#181;L/mouse) with a 25 G needle.</li>
		<li>After luciferase substrate administration, place the mice back in the isolation chamber with their chests facing up and nasal cavity inside the manifold cone to keep the animal anesthetized during the imaging procedure.</li>
		<li>Transfer the isolation chamber to the IVIS machine, and instantaneously after closing the IVIS imager door, select Acquire in the software program to initialize (Figure 2A).</li>
		<li>To analyze bioluminescence images acquired, utilize the ROI (region of interest) tool in the software to designate the precise signal and measure the flux (Figure 2B).</li>
		<li>Click on Measure and allow the system to begin assessing the bioluminescence in photons to provide the absolute photon emission comparable to the output measurements provided by the various parameters.</li>
		<li>Image mice at 1-, 2-, 4- and 6-days post-infection.</li>
		<li>After&#160;imaging&#160;place mice back into cage.</li>
	</ol>
	</li>
	<li>Fluorescence analysis in K18 hACE2 transgenic mice infected with rSARS-CoV-2/Venus (Figure 3 and Figure 4) 
	NOTE: This experiment follows the schematic representation and rSARS-CoV-2 shown in Figure 3. See Table of Materials for details instruments and systems required.
	<ol>
		<li>Initiate the imaging software and set up the parameters, set excitation (500 nm) and emission filters (530 nm), click image mode to Fluorescence and set the exposure time to auto.</li>
		<li>Intraperitoneally euthanize the mice using a lethal dose of Fatal-Plus (&#62;100 mg/kg). Following euthanasia, disinfect the incision site with 70% ethanol.</li>
		<li>Using tweezers, pull the skin, make an incision from the sternum to the abdomen and cut the incision from the sides with scissors. Cut the hepatic vein to minimize the amount of blood in the lungs and avoid high background signals during imaging.</li>
		<li>Using scissors, cut the sternum and open the ribcage. Next, snip the end of the trachea with scissors and remove the lungs with tweezers.</li>
		<li>Place the excised lungs in a 6-well plate containing 2 mL of 1x PBS and rinse to remove excess blood. Minimize the possibility of contamination between samples by disinfecting and cleaning surgery tools between samples using 70% ethanol.</li>
		<li>After initializing the IVIS machine, place the lungs in a black tray and separate the tissues from each other.</li>
		<li>Place the tray inside the isolation chamber inside the biosafety cabinet, and then transfer the isolation chamber to the IVIS. Close the door and click on Acquire to initiate the imaging system (Figure 4A).</li>
		<li>To analyze fluorescence, utilize the ROI tool and draw ROIs around each of the individual lungs. Measure each ROI manually and then use the average radiant efficiency values given and subtract from those of the mock-infected mice (Figure 4B).</li>
		<li>Once imaging is complete, place the tissues on ice for same-day analysis or in a cryotube for dry ice freezing to store at -80 &#176;C for later processing.</li>
	</ol>
	</li>
	<li>Lung bright field imaging and pathology scoring of K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 2A-C) and rSARS-CoV-2/Venus (Figure 4A-C)
	<ol>
		<li>After imaging bioluminescence of mice infected with rSARS-CoV-2/Nluc, rSARS-CoV-2/WT, and mock-infected, return the mice to their cages. Proceed with euthanasia and the ex vivo bright field imaging of mice lungs (Figure 2A).</li>
		<li>After imaging ex vivo fluorescence of lungs of infected mice, take bright-field images of the lungs (Figure 4A).</li>
		<li>Analyze the gross lesions on the surface of the lung using ImageJ (Figures 2C and 4C).</li>
		<li>Open the lung image to be analyzed in ImageJ.</li>
		<li>Calculate the ratio of pixel to cm, use the Straight tool and measure the actual length of the image.</li>
		<li>After selecting, click on Analyze &#62; Measure to calculate the length of pixels for the length.</li>
		<li>Click on Analysis &#62; Set Scale and input the numbers calculated from the previous step.</li>
		<li>Use the Freehand Selections tool and select the entire lung surface. Click on Analyze &#62; Measure to measure total lung area.</li>
		<li>Click on Edit &#62; Selection &#62; Make Inverse to select the rest of the lung area, then press delete or backspace to remove the background.</li>
		<li>Remove the selected area, then click on Image &#62; Adjust &#62; Color Threshold.</li>
		<li>To select pathologic lesion area, adjust Brightness to a minimum (between 1 to 50) and a maximum (between 50 to 200), depending on the levels of congestion and hemorrhages present.</li>
		<li>Once pathologic lesions are selected, click on Select at the bottom of the threshold color panel, then click on Analyze &#62; Measure to measure pathologic areas.</li>
		<li>Calculate the percentage of gross lesions with the formula: % of pathologic area = (measurement of pathologic area/total lung surface) x 100.</li>
	</ol>
	</li>
	<li>Viral titrations (Figure 2D and Figure 4D)
	<ol>
		<li>Upon imaging, complete the mice euthanasia and collect lungs, brain, and nasal mucosa.</li>
		<li>Place the lungs, brain, and nasal mucosa into separate sterile tissue homogenizers and add 1 mL of cold 1x PBS.</li>
		<li>Homogenize the samples by centrifuging at 21,500 x g for 10 min to pellet cell debris. Collect and transfer the supernatants into a new sterile tube and discard the pellet.</li>
		<li>If viral titrations are performed the same day, store the supernatants at 4 &#176;C. Alternatively, freeze the supernatant of the homogenized samples at -80 &#176;C until being evaluated later.</li>
		<li>Utilizing the supernatants obtained from the tissue homogenates make 10-fold serial dilutions and infect confluent monolayers of Vero E6 cells with 1 mL of each dilution of the supernatant (6-well plate format, 1.2 &#215; 106 cells/well, triplicates).</li>
		<li>Let the virus adsorb for 1 h at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>After viral adsorption, wash the cells with 1 mL of 1x PBS and incubate in 2 mL of post-infection media containing 1% Agar in the humidified 5% CO2 incubator at 37 &#176;C for 72 h.</li>
		<li>After the incubation, inactivate the plates in 10% neutral buffered formalin for 24 h at 4 &#176;C, ensure the entire plate is submerged.</li>
		<li>Take plates out of BSL3 and wash the cells three times with 1 mL of 1x PBS and permeabilize with 1 mL of 0.5% Triton X-100 for 10 min at room temperature (RT).</li>
		<li>Block the cells with 1 mL of 2.5% bovine serum albumin (BSA) in 1x PBS for 1 h at 37 &#176;C, followed by incubation in 1 mL of 1 &#181;g/mL of the SARS-CoV nucleocapsid (N) protein cross-reactive monoclonal antibody (1C7C7), diluted in 2.5% BSA for 1 h at 37 &#176;C.</li>
		<li>Wash the cells three times with 1 mL of 1x PBS and develop the plaques using the ABC kit and DAB Peroxidase Substrate kit according to the manufacturers&#39; instructions.</li>
		<li>Calculate the viral titers as PFU/mL. 
		&#8203;NOTE: Calculate with the formula PFU/mL = dilution factor x number of plaques x (1 mL/inoculum volume).</li>
	</ol>
	</li>
	<li>Nluc activity in tissues of K18 hACE2 transgenic mice infected with rSARS-CoV-2/Nluc (Figure 2E)
	<ol>
		<li>Quantify the presence of Nluc in the organ homogenates from mock, rSARS-CoV-2/WT, and rSARS-CoV-2/Nluc infected K18 hACE2 transgenic mice using a luciferase assay following the manufacturers&#39; instructions.</li>
	</ol>
	</li>
	<li>Evaluation of morbidity and mortality (Figure 2F, G and Figure 4E, F)
	<ol>
		<li>Intranasally infect 4-6-week-old female K18 hACE2 transgenic mice with 105 PFU of rSARS-CoV-2/WT, rSARS-CoV-2/Venus, rSARS-CoV-2/Nluc, or mock-infected as described in section 3.1.</li>
		<li>Monitor and weigh the mice over 12 days at the same time to minimize the weight variation due to food ingestion. Euthanize the mice that lose 25% of their initial body weight since they have reached a humane endpoint and note these mice as succumbing to viral infection.</li>
		<li>After 12 days, euthanize the mice that survive viral infection and calculate the % of body weight change and survival.</li>
	</ol>
	</li>
</ol>

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K., Noguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bioluminescence+imaging.">Cellular bioluminescence imaging.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Tran, V., Moser, L. A., Poole, D. S., Mehle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+real-time+in+vivo+imaging+of+an+influenza+reporter+virus+reveals+dynamics+of+replication+and+spread.">Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.</a> Journal of Virology. 87 (24), 13321-13329 (2013).</li><li>Schoggins, J. W., 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=Dengue+reporter+viruses+reveal+viral+dynamics+in+interferon+receptor-deficient+mice+and+sensitivity+to+interferon+effectors+in+vitro.">Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (36), 14610-14615 (2012).</li><li>Luker, G. D., 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=Noninvasive+bioluminescence+imaging+of+herpes+simplex+virus+type+1+infection+and+therapy+in+living+mice.">Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice.</a> Journal of Virology. 76 (23), 12149-12161 (2002).</li><li>Li, X., 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=Development+of+a+rapid+antiviral+screening+assay+based+on+eGFP+reporter+virus+of+Mayaro+virus.">Development of a rapid antiviral screening assay based on eGFP reporter virus of Mayaro virus.</a> Antiviral Research. 168, 82-90 (2019).</li><li>Kirui, J., Freed, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+validation+of+a+highly+sensitive+bioluminescent+HIV-1+reporter+vector+that+simplifies+measurement+of+virus+release.">Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release.</a> Retrovirology. 17 (1), 12 (2020).</li><li>Shang, B., 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=Development+and+characterization+of+a+stable+eGFP+enterovirus+71+for+antiviral+screening.">Development and characterization of a stable eGFP enterovirus 71 for antiviral screening.</a> Antiviral Research. 97 (2), 198-205 (2013).</li><li>Zou, G., Xu, H. Y., Qing, M., Wang, Q. Y., Shi, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+stable+luciferase+dengue+virus+for+high-throughput+screening.">Development and characterization of a stable luciferase dengue virus for high-throughput screening.</a> Antiviral Research. 91 (1), 11-19 (2011).</li><li>Ye, C., 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=Rescue+of+SARS-CoV-2+from+a+single+bacterial+artificial+chromosome.">Rescue of SARS-CoV-2 from a single bacterial artificial chromosome.</a> mBio. 11 (5), 02168 (2020).</li><li>Avila-Perez, G., Park, J. G., Nogales, A., Almazan, F., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+recombinant+Zika+virus+from+a+bacterial+artificial+chromosome+cDNA+clone.">Rescue of recombinant Zika virus from a bacterial artificial chromosome cDNA clone.</a> Journal of Visualized Experiments: JoVE. (148), e59537 (2019).</li><li>Chiem, 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=A+bifluorescent-based+assay+for+the+identification+of+neutralizing+antibodies+against+SARS-CoV-2+variants+of+concern+in+vitro+and+in+vivo.">A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo.</a> Journal of Virology. , (2021).</li><li>Committee for the update of the guide for the care and use of laboratory animals., Institute for laboratory animal research (U.S) &amp; National Academies Press (U.S.). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+for+the+care+and+use+of+laboratory+animals.+8th+edn.">Guide for the care and use of laboratory animals. 8th edn.</a> National Research Council (US). , (2011).</li><li>Ye, C., 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=Analysis+of+SARS-CoV-2+infection+dynamic+in+vivo+using+reporter-expressing+viruses.">Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (41), (2021).</li></ol>

The authors declare that the research was conducted in the absence of any commercial, financial and non-financial, or personal conflict of interest in relation to the work described.

This protocol demonstrates the feasibility of using these rSARS-CoV-2 expressing reporter genes to monitor viral infections in vivo. Both reporter-expressing recombinant viruses provide an excellent tool for studying SARS-CoV-2 infections in vivo. The described ex vivo (rSARS-CoV-2/Venus) and in vivo (rSARS-CoV-2/Nluc) imaging systems represent an excellent option to understand the dynamics of SARS-CoV-2 infection, viral pathogenesis and to identify infected cells/organs at different t...

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus that belongs to the Betacoronavirus lineage in the Coronaviridae family1. This viral family is divided into Alpha-, Beta-, Gamma-, and Delta-coronavirus1. Alpha- and Betacoronaviruses mainly infect mammals, whereas Gamma- and Deltacoronavirus infect almost exclusively birds2. To date, seven coronaviruses (CoV) have crossed species barriers and emerged as human coronaviruses (HCoV): two alpha-CoVs (HCoV-229E and HCoV-NL63) and five beta-CoVs (HCoV-OC43, HCoV-HKU1, SARS-CoV, Middle East respiratory syndrome coronavirus [MERS-CoV], and SARS-CoV-2)3,4,5,6. SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic, causing severe lower respiratory tract infection7. Prior to the emergence of SARS-CoV-2, there were two epidemic outbreaks caused by CoVs: SARS-CoV in Guangdong Providence, China, from 2002-2003, with a case fatality rate (CFR) of about 9.7%; and MERS-CoV in the Middle East from 2012 to present, with a CFR of about 34%7,8. SARS-CoV-2 has an overall CFR between 3.4%-49%, with underlying conditions contributing to a higher CFR8,9. Since its discovery in December 2019, in Wuhan, China, SARS-CoV-2 has been responsible for over 242 million human infections and more than 4.9 million human deaths worldwide7,10,11,12. Notably, since late 2020, new SARS-CoV-2 variants of concern (VoC) and variants of interest (VoI) have impacted virus characteristics, including transmission and antigenicity9,13, and the overall direction of the COVID-19 pandemic. For the treatment of SARS-CoV-2 infections, there is currently only one United States (U.S.) Food and Drug Administration (FDA) therapeutic antiviral (remdesivir) and one Emergency Use Authorization (EUA) drug (baricitinib, to be administered in combination with remdesivir)14. There are also 6 approved EUA monoclonal antibodies: REGEN-COV (casirivimab and imdevimab, administered together), sotrovimab, tocilizumab, and bamlanivimab and etesevimab administered together15,16,17,18,19. There is currently only one FDA-approved prophylactic vaccine, Pfizer-BioNTech, and two other prophylactic vaccines (Moderna and Janssen) have been EUA approved20,21,22,23,24. However, with the uncontrolled infection rate and the emergence of VoC and VoI, SARS-CoV-2 still poses a threat to human health. Therefore, new approaches are urgently needed to identify efficient prophylactics and therapeutics to control SARS-CoV-2 infection and the still ongoing COVID-19 pandemic.
Studying SARS-CoV-2 requires laborious techniques and secondary approaches to identify the presence of the virus in infected cells and/or validated animal models of infection. The use of reverse genetics has allowed for the generation of recombinant viruses to answer important questions in the biology of viral infections. For instance, reverse genetics techniques have provided means to uncover and understand the mechanisms of viral infection, pathogenesis, and disease. Likewise, reverse genetics approaches have paved the way to engineer recombinant viruses lacking viral proteins to understand their contribution in viral pathogenesis. In addition, reverse genetics techniques have been used to generate recombinant viruses expressing reporter genes for in vitro and in vivo applications, including identifying prophylactic and/or therapeutic approaches for the treatment of viral infections. Fluorescent and bioluminescent proteins are the most commonly used reporter genes due to their sensitivity, stability, and easy detection based on the improvement of new technologies25,26. In vitro, fluorescent proteins have been shown to serve as a better option for the localization of viruses in infected cells, while luciferases are more convenient for quantification studies27,28,29. In vivo, luciferases are preferred over fluorescent proteins for whole animal imaging, while fluorescent proteins are preferred for the identification of infected cells or ex vivo imaging30,31,32. The use of reporter-expressing recombinant viruses has served as a powerful tool for the study of viruses in many families, including, among others, flaviviruses, enteroviruses, alphaviruses, lentiviruses, arenaviruses, and influenza viruses28,33,34,35,36.
To overcome the need for secondary approaches to study SARS-CoV-2 and characterize real-time SARS-CoV-2 infection in vivo, we have generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins using our previously described bacterial artificial chromosomes(BAC)-based reverse genetics, which are maintained as a single copy in E. coli in order to minimize toxicity of virus sequences during its propagation in bacteria37,38. Notably, rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus showed rSARS-CoV-2/WT-like pathogenicity in vivo. The high level of Venus expression from rSARS-CoV-2/Venus allowed detecting viral infection in the lungs of infected K18 hACE2 transgenic mice using an in vivo imaging system (IVIS)39. The levels of Venus expression correlated well with viral titers detected in the lungs, demonstrating the feasibility of using Venus expression as a valid surrogate of SARS-CoV-2 infection. Using rSARS-CoV-2/Nluc, we were able to track the dynamics of viral infection in real-time and longitudinally assess SARS-CoV-2 infection in vivo using the same IVIS approach in K18 hACE2 transgenic mice.

<table><tbody><tr><td>0.5% Triton X-100</td><td>J.T.Baker</td><td>X198-07</td><td>Store at room temperature (RT)</td></tr><tr><td>1% DEAE-Dextran</td><td>MP Biomedicals</td><td>195133</td><td></td></tr><tr><td>10% Formalin solution, neutral buffered</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Agar</td><td>Oxoid</td><td>LP0028</td><td></td></tr><tr><td>24-well Cell Culture Plate</td><td>Greiner Bio-one</td><td>662160</td><td></td></tr><tr><td>5% Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S-5761</td><td></td></tr><tr><td>6-well Cell Culture Plate</td><td>Greiner Bio-one</td><td>657160</td><td></td></tr><tr><td>96-well Cell Culture Plate</td><td>Greiner Bio-one</td><td>655-180</td><td></td></tr><tr><td>African green monkey kidney epithelial cells (Vero E6)</td><td>ATCC</td><td>CRL-1586</td><td></td></tr><tr><td>Ami HT</td><td>Spectral Instruments Imaging</td><td></td><td></td></tr><tr><td>Aura Imaging Software 3.2.0</td><td>Spectral Instruments Imaging</td><td></td><td>Image analysis software</td></tr><tr><td>Bovine Serum Albumin (BSA), 35%</td><td>Sigma-Aldrich</td><td>A9647</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Cell culture grade water</td><td>Corning</td><td>25-055-CV</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Corning Cellgro</td><td>15-013-CV</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Anesthesia gas machine</td><td>Veterinary Anesthesia Systems, Inc.</td><td>VAS 2001R</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-050</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Four- to six-week-old female K18-hACE2 transgenic mice</td><td>The Jackson Laboratory</td><td>34860</td><td></td></tr><tr><td>Graphpad Prism Version 9.1.0</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>1001936040</td><td>Store at RT</td></tr><tr><td>MARS Data Analysis Software</td><td>BMG LABTECH</td><td></td><td></td></tr><tr><td>MB10 tablets</td><td>QUIP Laboratories</td><td>MBTAB1.5</td><td>Store at RT</td></tr><tr><td>Nano-Glo Luciferase Assay Reagent</td><td>Promega</td><td>N1110</td><td>This reagent is used to measure Nluc activity. Store at -20 &amp;deg;C</td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>ThermoFisher Scientific</td><td>269620</td><td></td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>ThermoFisher Scientific</td><td>269620</td><td></td></tr><tr><td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td><td>Corning</td><td>30-009-CI</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>PHERAstar FSX</td><td>BMG LABTECH</td><td>PHERAstar FSX</td><td></td></tr><tr><td>Precelleys Evolution homogenizer</td><td>Bertin Instruments</td><td>P000062-PEVO0-A</td><td></td></tr><tr><td>Soft tissue homogenizing CK14 &amp;ndash; 7 mL</td><td>Bertin Instruments</td><td>P000940-LYSK0-A</td><td></td></tr><tr><td>T75 EasYFlask</td><td>ThermoFisher Scientific</td><td>156499</td><td></td></tr><tr><td>VECTASTAIN ABC-HRP Kit, Peroxidase</td><td>Vector Laboratories</td><td>PK-4002</td><td>ABC kit and DAB Peroxidase Substrate kit</td></tr></tbody></table>

live imaging and quantification of viral infection in k18 hace2 transgenic mice using reporter-expressing recombinant sars-cov-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

rSARS-CoV-2/Nluc infection in K18 hACE2 transgenic mice (Figures 1 and 2) 
Figure 1A shows a schematic representation of the rSARS-CoV-2/WT (top) and rSARS-CoV-2/Nluc (bottom) used to assess infections in vivo. Figure 1B shows the schematic flow chart applied to assess rSARS-CoV-2/Nluc infection dynamics in K18 hACE2 transgenic mice. Four-to-six-week-old female K18 hACE2 transgenic mic...

Watch this Scientific Journal Video about Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2 at JoVE.com

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

live-imaging-quantification-viral-infection-k18-hace2-transgenic-mice

Research

JoVE Journal

Immunology and Infection

2.6K Views.  Texas Biomedical Research Institute. This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Video: Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Cancer Research

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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, ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

Neuroscience

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Biology

AAV-Based Therapies via Intravenous Injection in Preclinical Mouse Models

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse&#39;s lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose.
Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Author Spotlight: Developing Gene Therapies for Muscular Dystrophies and Neuromuscular Disorders

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...

Medicine

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

Source: Chiem, K., et al. <a href="https://app.jove.com/v/59890">A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.</a> J. Vis. Exp. (150), (2019).
This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...