This video describes the use of whole body bioluminescent imaging for the study of bacterial trafficking in live mice, with an emphasis on detection of bacterial growth within tumor xenografts bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors, following intravenous administration growing either external to or within tumor cells. Bacterial strains engineered to express the lux gene cassette facilitate detection of the bacteria and concurrently tumors. The value of non-invasive longitudinal monitoring of bacterial strains in vivo is well accepted, reducing animal usage and generating more statistically relevant data than endpoint assays.
This procedure involves the induction of subcutaneous tumors in mice. Tumors are permitted to develop and monitored over time. Bacteria are Administered and mice are subjected to bioluminescence imaging at dedicated time points For the Experiments described here.
Subcutaneous tumors are induced in mice of either an immune competent or a thymic background. Depending on choice of xenograft, Lux type bacteria of the desired species such as e coli, Are grown as overnight cultures. Bacterial cells are washed and resuspended at the desired concentration for intravenous administration.
10 to the six. Bacterial cells in an injection volume of 100 microliters is routinely used. Mice bearing subcutaneous tumors routinely 100 millimeters Cuban volume are prepared for treatment.
Using a 29 gauge syringe needle, the bacterial suspension is injected to the lateral tail vein. The bacteria Preferentially replicated within the tumor mass over time. Intratumoral bacteria continue to replicate for several weeks and are detectable by IVUS Imaging between three and six days.
Post administration For bioluminescent imaging live and anesthetized mice are placed within the 37 degree chamber of the ivus. If desired, several mice can be imaged Simultaneously. Photographic and luminescent light images are collected using a cooled CCD camera at the desired software settings.
Depending on the imaging device, two or three dimensional analysis may be performed. A robust And linear relationship between intratumoral bacterial numbers and the levels of light produced is achieved permitting. Reliable estimation of bacterial Numbers in vivo LX expressing bacteria May also be detected from deep within the body.
Lux expressing gut colonizing commensal bacteria are visualized for several weeks. Post cavage administration further demonstrating the utility of bioluminescent imaging for the analysis of lux expressing bacteria within research animals.
