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首页> 外文会议>Conference on Multiphoton Microscopy in the Biomedical Sciences III Jan 26-28, 2003 San Jose, California, USA >Optical Gene Transfer by Femtosecond Laser Pulses
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Optical Gene Transfer by Femtosecond Laser Pulses

机译：飞秒激光脉冲进行光学基因转移

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摘要

Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (10~(12) W/cm~2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturised cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany) and focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.
机译：细胞的靶向转染是基因治疗和相关生物医学应用的重要技术。我们描述了高强度（10〜（12）W / cm〜2）近红外（NIR）80 MHz纳米焦耳飞秒激光脉冲如何在微小焦距内产生高度局部化的膜穿孔，从而实现哺乳动物的非侵入性直接转染DNA细胞。我们将中国仓鼠卵巢（CHO），大鼠袋鼠肾上皮细胞（PtK2）和大鼠成纤维细胞悬浮于0.5 ml无菌微型细胞室（JenLab GmbH，Jena，Germany）中，该细胞室含有0.2μg质粒DNA载体pEGFP-N1（4.7 kb），它编码绿色荧光蛋白（GFP）。 NIR激光束被引入飞秒激光扫描显微镜（JenLab GmbH，德国耶拿），并聚焦在靶细胞的细胞膜边缘16毫秒。通过使用与时间相关的单光子计数的双光子荧光寿命成像，原位评估EGFP的整合和表达效率。安全有效地将外源DNA转移到特定细胞类型（包括干细胞）中的独特能力，可以避免机械，电气或化学手段，将具有许多应用，例如靶向基因治疗和DNA疫苗接种。

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