CAREER: Optics in Biological Research, Education, and Outreach

Schaffer：该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。PI建议开发工具，将外源DNA引入活体啮齿动物大脑中特定目标细胞，为细胞靶向基因操作打开大门。这一新能力将把基因操纵技术的力量扩展到一类新的实验，在这种实验中，单细胞靶向能够在体内研究细胞与细胞的相互作用和疾病的早期阶段。他们将使用紧密聚焦的飞秒激光脉冲来消融目标细胞膜上的一个小的、瞬时的洞，允许已经注入细胞外空间的DNA进入细胞。这个洞很快就会重新密封，让细胞活着并发挥作用。初步数据证实了靶细胞在体外和体内的光学作用，以及光学转基因DNA的体外表达。他们将把这项技术扩展到体内靶向脑细胞的转基因。他们进一步建议使用这项技术来表达单个脑神经元中的光门离子通道，并使用细胞分辨钙成像来跟踪光学启动的转基因细胞的激发如何影响周围细胞的活动。这项工作可能为在活体内研究皮质神经元回路的功能连接提供一种独特的手段。

0846534Schaffer"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."The PI proposes to develop tools to enable the introduction of exogenous DNA into specifically targeted cells in the brain of a live rodent, opening the door to cell-targeted genetic manipulation. This new capability will extend the power of genetic manipulation techniques to a new class of experiments, where single-cell targeting enables cell-cell interactions and early stages of disease to be studied, in vivo. They will use tightly-focused femtosecond laser pulses to ablate a small, transient hole in the membrane of a targeted cell, allowing DNA that has been injected into the extracellular space to enter the cell. This hole rapidly reseals, leaving the cell alive and functional. Preliminary data confirms optoporation of targeted cells in vitro and in vivo as well as in vitro expression of optically transfected DNA. They will extend this technique to in vivo transfection in targeted brain cells. They further propose to use this technology to express light-gated ion channels in single brain neurons and use cell-resolved calcium imaging to follow how the optically initiated firing of the transfected cell effects activity in surrounding cells. This work could provide a unique means to study the functional connectivity of cortical neuronal circuits, in vivo.