Kimmel Center for Biology & Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Ave, New York, NY 10016, USA.
Circ Res. 2012 Mar 30;110(7):938-47. doi: 10.1161/CIRCRESAHA.111.254375. Epub 2012 Feb 28.
The formation and maintenance of a functional vasculature is essential for normal embryonic development, and genetic changes that affect the vasculature underlie pathogenesis in many human diseases. In vivo imaging in mouse models is required to understand the full complexity of mammalian vascular formation, which is a dynamic and 3-dimensional process. Optical microscopy of genetically expressed fluorescent reporter proteins offers high resolution but limited depth of penetration in vivo. Conversely, there are a plethora of molecular probes for alternative in vivo vascular imaging modalities, but few options for genetic control of contrast enhancement.
To develop a reporter system for multimodal imaging of genetic processes involved in mammalian vascular biology.
To approach this problem, we developed an optimal tagging system based on Biotag-BirA technology. In the resulting Biotag reporter system, coexpression of 2 interacting proteins results in biotin labeling of cell membranes, thus enabling multimodal imaging with "avidinated" probes. To assess this approach for in vivo imaging, we generated transgenic mice that expressed the Biotag-BirA transgene from a minimal Tie2 promoter. A variety of imaging methods were used to show the utility of this approach for quantitative analysis in embryonic and adult models of vascular development, using intravascular injection of avidinated probes for near infrared, ultrasound, and magnetic resonance imaging.
The present results demonstrate the versatility of the Biotag system for studies of vascular biology in genetically engineered mice, providing a robust approach for multimodal in vivo imaging of genetic processes in the vasculature.
功能性血管的形成和维持对于正常胚胎发育至关重要，而影响血管的遗传变化是许多人类疾病发病机制的基础。为了了解哺乳动物血管形成的全部复杂性，需要在小鼠模型中进行体内成像，这是一个动态的、三维的过程。遗传表达荧光报告蛋白的光学显微镜提供了高分辨率，但体内穿透深度有限。相反，有大量的分子探针可用于替代体内血管成像模式，但用于遗传控制对比增强的选择很少。
开发用于哺乳动物血管生物学中遗传过程的多模式成像的报告系统。
为了解决这个问题，我们基于 Biotag-BirA 技术开发了一个最优标记系统。在由此产生的 Biotag 报告系统中，两种相互作用蛋白的共表达导致细胞膜的生物素标记，从而能够使用“亲和素化”探针进行多模式成像。为了评估这种方法在体内成像中的应用，我们生成了表达 Biotag-BirA 转基因的转基因小鼠，该转基因来自最小的 Tie2 启动子。使用多种成像方法表明，这种方法可用于血管发育的胚胎和成年模型中的定量分析，通过血管内注射亲和素化探针进行近红外、超声和磁共振成像。
本研究结果证明了 Biotag 系统在基因工程小鼠血管生物学研究中的多功能性，为血管遗传过程的多模式体内成像提供了一种强大的方法。
