The authors wish to acknowledge support relevant to this manuscript from the European Commission Seventh Framework Programme (PIOF-GA-2009-255466) and the Irish Health Research Board (HRA_POR/2010/138). Lux-tagged E. coli was a kind gift from Dr. Cormac Gahan, University College Cork.

1. Tumor Induction
<ol>
 <li>For routine tumor induction, the minimum tumorigenic dose of cells suspended in 200 &mu;l of serum-free culture medium was injected subcutaneously (s.c.) into the flank of infection free 6-8 week old female Balb/C or athymic MF1-nu/nu mice n=6 (Harlan, Oxfordshire, UK) (1 x 106 4T1 cells) using a 21-gauge syringe needle. The viability of cells used for inoculation was greater than 95 % as determined by visual count using a haemocytometer and Trypan Blue Dye Exclusion (Gibco). <...

<ol>
	<li>Cronin, M., 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=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Tangney, M., Ahmad, S., Collins, S. A., O'Sullivan, G. 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+therapy+for+prostate+cancer.">Gene therapy for prostate cancer.</a> Postgrad Med. 122, 166-180 (2010).</li><li>Morrissey, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+targeting+with+systemically+administered+bacteria.">Tumour targeting with systemically administered bacteria.</a> Curr. Gene Ther. 10, 3-14 (2010).</li><li>Collins, S. 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=Viral+vectors+in+cancer+immunotherapy:+which+vector+for+which+strategy.">Viral vectors in cancer immunotherapy: which vector for which strategy.</a> Curr. Gene Ther. 8, 66-78 (2008).</li><li>Yu, Y. A., Zhang, Q., Szalay, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+conditions+required+for+tumor+colonization+by+intravenously+delivered+bacteria.">Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria.</a> Biotechnol. Bioeng. 100, 567-578 (2008).</li><li>Baban, C. K., Cronin, M., O'Hanlon, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+as+vectors+for+gene+therapy+of+cancer.">Bacteria as vectors for gene therapy of cancer.</a> Bioeng. Bugs. 1, 385-394 (2010).</li><li>Cronin, M., 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=Orally+administered+bifidobacteria+as+vehicles+for+delivery+of+agents+to+systemic+tumors.">Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors.</a> Mol. Ther. 18, 1397-1407 (2010).</li><li>van Pijkeren, J. P., 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+novel+Listeria+monocytogenes-based+DNA+delivery+system+for+cancer+gene+therapy.">A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.</a> Hum. Gene Ther. 21, 405-416 (2010).</li><li>Ahmad, 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=Induction+of+effective+antitumor+response+after+mucosal+bacterial+vector+mediated+DNA+vaccination+with+endogenous+prostate+cancer+specific+antigen.">Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen.</a> J. Urol. 186, 687-693 (2011).</li><li>Riedel, C. U., 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=Improved+luciferase+tagging+system+for+Listeria+monocytogenes+allows+real-time+monitoring+in+vivo+and+in+vitro.">Improved luciferase tagging system for Listeria monocytogenes allows real-time monitoring in vivo and in vitro.</a> Appl Environ Microbiol. 73, 3091-3094 (2007).</li><li>Cheng, C. M., 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=Tumor-targeting+prodrug-activating+bacteria+for+cancer+therapy.">Tumor-targeting prodrug-activating bacteria for cancer therapy.</a> Cancer Gene Ther. 15, 393-401 (2008).</li><li>Foucault, M. L., Thomas, L., Goussard, S., Branchini, B. R., Grillot-Courvalin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+the+study+of+intestinal+colonization+by+Escherichia+coli+in+mice.">In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.</a> Appl. Environ. Microbiol. 76, 264-274 (2010).</li><li>Collins, S. A., Hiraoka, K., Inagaki, A., Kasahara, N., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+Imaging+For+Gene+&amp;+Cell+Therapy.">PET Imaging For Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li><li>Tangney, M., Francis, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Optical+Imaging+in+Gene+&amp;+Cell+Therapy.">In vivo Optical Imaging in Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li></ol>

In the context of gene therapy, the use of biological agents for delivery of therapeutic genes to patients has shown great promise 3-5. Like viruses, the innate biological properties of bacteria permit efficient DNA delivery to cells or tissues, particularly in the context of cancer. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally, either external to (non-invasive species) or within tumor cells (pathogens)....

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>4T1 cell line</td>
 <td>ATCC</td>
 <td>CRL-2539</td>
 <td>Syngeneic breast cancer model derived from a spontaneously arising BALB/c mammary tumor</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma-Aldrich</td>
 <td>D6429</td>
 <td>Dulbecco's Modified Eagle's Medium</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Sigma-Aldrich</td>
 <td>D8537</td>
 <td>Phosphate Buffered Saline</td>
 </tr>
 <tr>
 <td>Xenogen IVIS</td>
 <td>Caliper Life Sciences</td>
 <td>&nbsp;</td>
 <td>IVIS 100 for 2D imaging; IVIS Spectrum for 3D.</td>
 </tr>
 <tr>
 <td>Luria Broth Miller (LB)</td>
 <td>Sigma-Aldrich</td>
 <td>L2542</td>
 <td>Growth medium for E. coli</td>
 </tr>
 <tr>
 <td>Erythromycin</td>
 <td>Sigma-Aldrich</td>
 <td>E5389</td>
 <td>Antibiotic</td>
 </tr>
 <tr>
 <td>Streptomycin</td>
 <td>Sigma-Aldrich</td>
 <td>S9137</td>
 <td>Antibiotic </td>
 </tr>
 <tr>
 <td>MF1nu/nu mice</td>
 <td>Harlan (UK)</td>
 <td>069(nu)/070(nu/+) </td>
 <td>Hsd:Athymic Nude-Foxn1nu </td>
 </tr>
 <tr>
 <td>Balb/c mice</td>
 <td>Harlan (UK)</td>
 <td>066</td>
 <td>Haplotype:&nbsp;H-2d</td>
 </tr>
 <tr>
 <td>Gavage needle </td>
 <td>Vet-tech Solutions (UK)</td>
 <td>DE009</td>
 <td>22G x 38mm straight gavage needle </td>
 </tr>
 <tr>
 <td>Syringe for IV injection </td>
 <td>BD BioSciences</td>
 <td>309309 - 1 ml</td>
 <td>Insulin syringe with 28 G x &frac12; inch micro-fine IV needle.</td>
 </tr>
 <tr>
 <td>Syringe for tumor inoculation</td>
 <td>Braun</td>
 <td>9161376V</td>
 <td>Omnifix 26 G x &frac12; inch needle</td>
 </tr>
 </tbody>
</table>

bioluminescent bacterial imaging in vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...