CAREER: Optics in Biological Research, Education, and Outreach

0846534 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引入活啮齿动物大脑中的特定靶细胞，为细胞靶向遗传操作打开大门。这种新的能力将把遗传操作技术的力量扩展到一类新的实验中，在这种实验中，单细胞靶向能够在体内研究细胞间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在靶细胞的膜上消融一个小的，短暂的洞，允许已经注入细胞外空间的DNA进入细胞。这个洞迅速重新密封，使细胞存活并发挥功能。初步数据证实了体外和体内靶细胞的光穿孔以及光学转染DNA的体外表达。他们将把这项技术扩展到靶向脑细胞的体内转染。他们进一步提出使用这种技术来表达单个脑神经元中的光门控离子通道，并使用细胞分辨钙成像来跟踪转染细胞的光学启动发射如何影响周围细胞的活动。这项工作可以提供一个独特的手段来研究皮层神经元电路的功能连接，在体内。

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.