Name: handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses
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Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.

NOTE: The protocols used have been approved by our institutional Animal Research Ethics Board at McMaster University according to Canadian Council on Animal Care guidelines. Experiments were performed at the McMaster University Central Animal Facility.

1. Culturing LCRT Cells
<ol>
	<li>Culture LCRT cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. Mainta...

<ol>
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S., Blanco, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cotton+rat+sigmodon+hispidus+model+of+respiratory+syncytial+virus+infection.">The cotton rat sigmodon hispidus model of respiratory syncytial virus infection.</a> Curr Top Microbiol Immunol. 372, 347-358 (2013).</li><li>Papp, Z., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+immunity+in+the+respiratory+tract+and+protection+from+bovine+herpesvirus+type+1+infection+by+different+routes+of+immunization+with+recombinant+adenovirus.">Induction of immunity in the respiratory tract and protection from bovine herpesvirus type 1 infection by different routes of immunization with recombinant adenovirus.</a> Viral Immunol. 11 (2), 79-91 (1998).</li><li>Hughes, T. C. R., Lilley, C. E., Ponce, R., Kaufman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+analysis+of+an+oncolytic+herpesvirus+encoding+granulocyte-macrophage+colony+stimulating+factor+for+the+treatment+of+malignant+melanoma.">Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma.</a> Journal of Oncolytic Virotherapy. 3, 11-20 (2014).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+biology+of+bovine+herpesvirus+type+1+(BHV-1),+its+role+as+a+cofactor+in+the+bovine+respiratory+disease+complex+and+development+of+improved+vaccines.">A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines.</a> Anim Health Res Rev. 8 (2), 187-205 (2007).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+herpesvirus+type+1+(BHV-1)+is+an+important+cofactor+in+the+bovine+respiratory+disease+complex.">Bovine herpesvirus type 1 (BHV-1) is an important cofactor in the bovine respiratory disease complex.</a> Vet Clin North Am Food Anim Pract. 26 (2), 303-321 (2010).</li><li>Hushur, O., Takashima, Y., Matsumoto, Y., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+bovine+herpesvirus+1+(BHV-1)+growth+in+non-permissive+cells+beyond+the+expression+of+immediate+early+genes.">Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes.</a> J Vet Med Sci. 66 (4), 453-455 (2004).</li><li>Cuddington, B. P., Dyer, A. L., Workenhe, S. T., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+bovine+herpesvirus+type+1+infects+and+kills+breast+tumor+cells+and+breast+cancer-initiating+cells+irrespective+of+tumor+subtype.">Oncolytic bovine herpesvirus type 1 infects and kills breast tumor cells and breast cancer-initiating cells irrespective of tumor subtype.</a> Cancer Gene Ther. 20 (5), 282-289 (2013).</li><li>Cuddington, B. P., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permissiveness+of+Human+Cancer+Cells+to+Oncolytic+Bovine+Herpesvirus+1+Is+Mediated+in+Part+by+KRAS+Activity.">Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity.</a> J Virol. 88 (12), 6885-6895 (2014).</li><li>Small, E. 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=A+phase+I+trial+of+intravenous+CG7870,+a+replication-selective,+prostate-specific+antigen-targeted+oncolytic+adenovirus,+for+the+treatment+of+hormone-refractory,+metastatic+prostate+cancer.">A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer.</a> Mol Ther. 14 (1), 107-117 (2006).</li><li>Freytag, S. O., 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=Phase+I+study+of+replication-competent+adenovirus-mediated+double+suicide+gene+therapy+for+the+treatment+of+locally+recurrent+prostate+cancer.">Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer.</a> Cancer Res. 62 (17), 4968-4976 (2002).</li><li>Benjamin, R., Helman, L., Meyers, P., Reaman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I/II+dose+escalation+and+activity+study+of+intravenous+injections+of+OCaP1+for+subjects+with+refractory+osteosarcoma+metastatic+to+lung.">A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung.</a> Hum Gene Ther. 12 (12), 1591-1593 (2001).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), 3-5 (1994).</li><li>Tsai, J. C., Garlinghouse, G., McDonnell, P. J., Trousdale, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+animal+model+of+adenovirus-induced+ocular+disease.+The+cotton+rat.">An experimental animal model of adenovirus-induced ocular disease. The cotton rat.</a> Arch Ophthalmol. 110 (8), 1167-1170 (1992).</li><li>Ginsberg, H. 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=A+mouse+model+for+investigating+the+molecular+pathogenesis+of+adenovirus+pneumonia.">A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia.</a> Proc Natl Acad Sci U S A. 88 (5), 1651-1655 (1991).</li><li>Russell, S. J., Peng, K. W., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virotherapy.">Oncolytic virotherapy.</a> Nat Biotechnol. 30 (7), 658-670 (2012).</li><li>Mossman, K. L., Saffran, H. A., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+mutants+are+hypersensitive+to+interferon.">Herpes simplex virus ICP0 mutants are hypersensitive to interferon.</a> J Virol. 74 (4), 2052-2056 (2000).</li><li>Mossman, K. L., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+and+ICP34.5+counteract+distinct+interferon-induced+barriers+to+virus+replication.">Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.</a> J Virol. 76 (4), 1995-1998 (2002).</li><li>Hummel, J. L., Safroneeva, E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ICP0-Null+HSV-1+and+interferon+signaling+defects+in+the+effective+treatment+of+breast+adenocarcinoma.">The role of ICP0-Null HSV-1 and interferon signaling defects in the effective treatment of breast adenocarcinoma.</a> Mol Ther. 12 (6), 1101-1110 (2005).</li><li>Papp, Z., Middleton, D. M., Mittal, S. K., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunization+with+recombinant+adenoviruses:+induction+of+immunity+and+protection+of+cotton+rats+against+respiratory+bovine+herpesvirus+type+1+infection.">Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.</a> J Gen Virol. 78 (11), 2933-2943 (1997).</li><li>Papp, Z., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pre-existing+adenovirus-specific+immunity+on+immune+responses+induced+by+recombinant+adenovirus+expressing+glycoprotein+D+of+bovine+herpesvirus+type+1.">The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.</a> Vaccine. 17 (7-8), 933-943 (1999).</li><li>Mittal, S. 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=Induction+of+systemic+and+mucosal+immune+responses+in+cotton+rats+immunized+with+human+adenovirus+type+5+recombinants+expressing+the+full+and+truncated+forms+of+bovine+herpesvirus+type+1+glycoprotein+gD.">Induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1 glycoprotein gD.</a> Virology. 222 (2), 299-309 (1996).</li><li>Steel, J. 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=Syngeneic+Cotton+Rat+Cancer+Model+for+Replicating+Adenoviral+Vectors.">Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors.</a> Molecular Therapy. 13 (1), 123 (2006).</li><li>Toth, 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=Cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Hum Gene Ther. 16 (1), 139-146 (2005).</li><li>Toth, K., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent,+semi-permissive+cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Methods Mol Med. 130, 157-168 (2007).</li><li>Steel, J. 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=Immunocompetent+syngeneic+cotton+rat+tumor+models+for+the+assessment+of+replication-competent+oncolytic+adenovirus.">Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus.</a> Virology. 369 (1), 131-142 (2007).</li><li>Workenhe, S. T., 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=Immunogenic+HSV-mediated+oncolysis+shapes+the+antitumor+immune+response+and+contributes+to+therapeutic+efficacy.">Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.</a> Mol Ther. 22 (1), 123-131 (2014).</li><li>Sobol, P. T., 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=Adaptive+antiviral+immunity+is+a+determinant+of+the+therapeutic+success+of+oncolytic+virotherapy.">Adaptive antiviral immunity is a determinant of the therapeutic success of oncolytic virotherapy.</a> Mol Ther. 19 (2), 335-344 (2011).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), (1994).</li></ol>

Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in ani...

Oncolytic viruses (OV) selectively replicate in tumor cells by exploiting biochemical differences between normal and tumor cells 1,2. There are two types of OVs: those that do not require a mutation to achieve selective oncolysis, referred to as naturally occurring wild-type viruses and those which must be engineered to achieve selective oncolysis. The collection of mutations within a given tumor type determines the nature of the selective growth advantage over normal cells for an OV 2. The safety and benefit of using OVs has been demonstrated in clinical trials 3-7. Despite advancements in the field of oncolytic virotherapy there exist gaps between pre-clinical and clinical results, suggesting that better models are needed to evaluate the antitumor efficacy of OVs.
Bovine herpesvirus type 1 (BHV-1) is a member of the Herpesviridae family, and Alphaherpesviridae subfamily. BHV-1 initiates bovine respiratory disease complex in cattle, manifesting in a wide variety of symptoms resembling a bad cold 8,9. BHV-1 binds attachment and entry receptors used by HSV-1, such as heparan sulfate and nectin-1 10. However, it binds CD155 in the place of nectin-2 10. BHV-1 has a very narrow host range such that it is unable to efficiently enter and initiate replication in normal and transformed murine cells 3,4,10. This makes the use of conventional murine models problematic. The oncolytic capacity of BHV-1 has been demonstrated in vitro11,12. BHV-1 has been shown to initiate replication in and kill human tumor cells from a variety of histological origins, including breast cancer cells and breast cancer initiating cells 11,12. However, the antitumor capacity of BHV-1 must be evaluated in vivo within the context of an immunocompetent host.
Human Adenovirus (Ad), for which there are 57 identified serotypes, most commonly causes respiratory illness in humans. Oncolytic Ad vectors have been evaluated for their antitumor efficacy with several advancing into clinical trials 13-15. Despite promising pre-clinical data, clinical results have fallen short of expectations. Human tumor xenograft models are typically used to study the antitumor efficacy of Ad vectors, although they exhibit attenuated immune responses to the virus 16,17. Furthermore, syngeneic murine models are non-permissive to Ad infection, making the evaluation of host immune responses using these models impractical 17,18.
The host immune system has been identified as the most influential mechanism by which OVs elicit tumor cell death 19. Antitumor responses between tolerized and non-tolerized tumor-associated antigen (TAA) models differ and can greatly impact the success of OV therapy. The HSV-1 OV KM100 (ICP0n212VP16in181420)20,21 elicited tumor regression in 80% of tumor-bearing mice in a murine Polyoma Middle T antigen mammary cancer model 22. However, in HER-2/neu models, the antitumor efficacy of KM100 varied between 20% complete regression in syngeneic mice and tumor stasis in transgenic, HER2-tolerized mice. Together these data highlight the importance of fully evaluating OVs using animal models that best recapitulate the human immune landscape to fully understand what features determine therapeutic success.
The cotton rat (Sigmodon hispidus), indigenous to North and South America, is most commonly used as a model of respiratory syncytial virus infection (as reviewed in 5). Cotton rats are also used in anti-BHV-1 vaccination research as they recapitulate the pathology associated with bovine respiratory disease complex 6,23. Furthermore, BHV-1 infection of cotton rats is immunogenic, inducing sustained mucosal and systemic immune responses 6,23-25. Cell lines have been derived from spontaneous fibrosarcoma and osteosarcomas of the mammary gland (LCRT) and bone (CCRT and VCRT), respectively 26. Cotton rats have been used to evaluate the in vivo efficacy of oncolytic Ad vectors as they are susceptible to Ad infection and exhibit similar pathology to humans 27-29. The use of immunocompromised models for the pre-clinical evaluation of OVs are not only less indicative of clinical responses to therapy but they fail to take into account the role of the immune system in oncolytic virotherapy 30,31. Therefore, the syngeneic and tumor-tolerized cotton rat models of mammary carcinoma and osteosarcoma are relevant models in which to evaluate the pre-clinical efficacy of OVs, such as BHV-1 and Ad which cannot be studied using conventional murine models.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>May use any brand&#160;</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline&#160;</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab, filter to sterilize</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>Gibco</td>
			<td>25030164</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Quality Biological Inc.</td>
			<td>110-001-101HI</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>T-150cm2 tissue culture flask</td>
			<td>Fisher Scientific</td>
			<td>14-826-80</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>1X TypLE Express</td>
			<td>Life Technologies</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate, flat bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td>May use any brand, must be tissue culture treated</td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>May use an alternative reagent for determination of cell viability</td>
		</tr>
		<tr>
			<td>8640 Teklad 22/5 Rodent diet</td>
			<td>Harlan&#160;</td>
			<td>8640</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8&#8221; corncob rodent bedding</td>
			<td>Harlan</td>
			<td>7092</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestlets</td>
			<td>Ancare</td>
			<td>-</td>
			<td>Made of pulped virgin cotton fiber, dust-free and autoclavable</td>
		</tr>
		<tr>
			<td>50 mL Conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>May use any brand, must be sterile</td>
		</tr>
		<tr>
			<td>Isoflurane USP, 99.9 %, inhalation anesthetic</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>M60302</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab</td>
		</tr>
		<tr>
			<td>10 % Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood)</td>
			<td>NAPCO</td>
			<td>Model used not currently available</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific Precision Heated Water Bath</td>
			<td>Fisher Scientific</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Reichert Bright-line Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Typhoon Trio BioAnalyzer&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Tecan Safire2 Multi-detection Microplate Reader</td>
			<td>Tecan</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Allegra 6R benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Table Top Anaesthesia machine</td>
			<td>VetEquip</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand, must be portable</td>
		</tr>
		<tr>
			<td>Wahl Peanut Mini Clippers</td>
			<td>Wahl</td>
			<td></td>
			<td>May use any brand of small clippers</td>
		</tr>
		<tr>
			<td>Insulin syringes 29 G x 1/2&#39;, 0.3 mL</td>
			<td>BD</td>
			<td>329464</td>
			<td>May use any brand. Insulin syringes are recommended as they make injections easier through the rat&#8217;s tough skin.&#160;</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>MedPro</td>
			<td>018-425</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>8940</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Dissecting Tissue Forceps</td>
			<td>Fisher Scientific</td>
			<td>13-812-41</td>
			<td>May use any brand</td>
		</tr>
	</tbody>
</table>

handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Due to the extremely excitable nature of cotton rats, being familiar with and utilizing procedures optimized to reduce the stress of the animals will ease in their use as a pre-clinical animal model. Use of proper handling techniques will also minimize risk to the researcher.
When using cotton rats it is imperative to stay calm. The rats are highly excitable and will attempt to escape their cage. Use of an enrichment tube and nestlets will minimize escape attempts. Figure 2 sh...

Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Research

JoVE Journal

Medicine

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