Danino Tal, Prindle Arthur, Hasty Jeff, Bhatia Sangeeta
Health Sciences and Technology, Massachusetts Institute of Technology.
J Vis Exp. 2013 Jul 6(77):e50540. doi: 10.3791/50540.
The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- (1)) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization(2). These strains specifically colonize tumors while remaining essentially non-virulent to the mouse(1). Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.
这些实验的目的是生成关于减毒鼠伤寒沙门氏菌在肿瘤内部生长的细菌菌落的生长和基因表达动态的定量时间进程数据。我们通过皮下注射人卵巢癌细胞系OVCAR-8（美国国立癌症研究所发展治疗部肿瘤库，马里兰州弗雷德里克）在小鼠体内生成了异种移植肿瘤模型。我们用组成型表达的荧光素酶（luxCDABE）质粒转化减毒鼠伤寒沙门氏菌菌株（ELH430:SL1344 phoPQ- (1)）以进行可视化(2)。这些菌株特异性地定殖于肿瘤，同时对小鼠基本无毒性(1)。一旦建立了可测量的肿瘤，就通过尾静脉以不同剂量静脉注射细菌。使用体内成像系统（IVIS）在60小时内实时监测肿瘤局部的细菌基因表达。在每个时间点，切除肿瘤，匀浆，并进行平板接种以定量细菌菌落，以便与基因表达数据进行关联。这些数据共同提供了对肿瘤内部生长的细菌的体内生长和基因表达动态的定量测量。
