Enhancement and optimization of plasmid expression in femtosecond optical transfection.
Enhancement and optimization of plasmid expression in femtosecond optical transfection.
复制标题
飞秒光转染中质粒表达的增强和优化。
DOI：
10.1002/jbio.201000105
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发表时间：
2011
影响因子：
2.8
通讯作者：
Praveen BB
中科院分区：
文献类型：
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作者：
Praveen BB
利用飞秒激光进行细胞转染越来越重要，因为它已被证明能够以无菌和相对非侵入的方式实现选择性转染。然而，由于许多因素，该技术的净效率受到限制，最终使其难以作为一种可行和广泛使用的技术使用。我们在这里报告了一种方法，以实现显著提高效率的飞秒光学转染。转染过程通过加入合适的含有核定位和DNA结合序列的合成肽来修改，以帮助DNA导入细胞核。我们用这种改进的方案实现了中国仓鼠卵巢（CHO‐K1）贴壁细胞转染效率的3倍增强。此外，在这种生化试剂的存在下，我们能够在不影响转染效率的情况下将所需的质粒浓度降低约70%。此外，我们首次报道了用修饰质粒转染胰蛋白酶化细胞后的高转染效率。这为高通量微流体光学转染装置的发展铺平了道路。（©2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim）
Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non‐invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3‐fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO‐K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ∼70% without compromising the transfection efficiency. Also, we report for the first time the successful photo‐transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)