Efficient gene delivery to primary alveolar epithelial cells by nucleofection
Efficient gene delivery to primary alveolar epithelial cells by nucleofection
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DOI：
10.1152/ajplung.00191.2013
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发表时间：
2013-12-01
影响因子：
4.9
通讯作者：
Vadasz, Istvan
中科院分区：
文献类型：
--
作者：
Grzesik, Benno A.;Vohwinkel, Christine U.;Vadasz, Istvan
Grzesik BA，Vohwinkel CU，Morty RE，Mayer K，赫罗尔德S，Seeger W，Vadasz I.通过核转染将基因有效递送至原代肺泡上皮细胞。Am J Physiol Lung Cell Mol Physiol 305：L786-L794，2013.首次发表于2013年9月27日; doi：10.1152/ajplung.00191.2013.-原代肺泡上皮细胞在肺研究中起着关键作用，特别是在关注气体交换、屏障功能和跨上皮转运过程时。然而，原代肺泡上皮细胞的有效转染仍然是一个重大挑战。在本研究中，我们应用核转染，一种新的方法，基因和寡核苷酸传递到细胞核的电穿孔，实现高效转染原代肺泡上皮II型（ATII）细胞。为了定量有效转染的ATII细胞的量，我们应用表达GFP的质粒并通过流式细胞术评估GFP表达细胞的量。对核转染的ATII细胞的分析显示，当使用3-8 μ g质粒DNA而不影响细胞活力时，浓度依赖性转染效率高达50%。培养的A549和H441细胞的核转染产生类似的转染率。重要的是，ATII细胞的核转染不干扰ATII单层的完整性，即使使用相对高浓度的质粒DNA。在随后的研究中，我们还通过核转染将小干扰RNA有效地递送到ATII细胞，从而沉默Akt和多配体受体megalin，其最近被证明在从肺泡腔中去除过量蛋白质中起关键作用，并有效地抑制megalin驱动的ATII细胞中白蛋白的摄取和跨细胞转运。因此，我们报告了通过核转染用质粒和寡核苷酸成功转染原代大鼠肺泡上皮细胞，具有高活力和一贯良好的转染率，而不损害细胞的关键生理特性。
Grzesik BA, Vohwinkel CU, Morty RE, Mayer K, Herold S, Seeger W, Vadasz I. Efficient gene delivery to primary alveolar epithelial cells by nucleofection. Am J Physiol Lung Cell Mol Physiol 305: L786-L794, 2013. First published September 27, 2013; doi:10.1152/ajplung.00191.2013.-Primary alveolar epithelial cells play a pivotal role in lung research, particularly when focusing on gas exchange, barrier function, and transepithelial transport processes. However, efficient transfection of primary alveolar epithelial cells continues to be a major challenge. In the present study, we applied nucleofection, a novel method of gene and oligonucleotide delivery to the nucleus of cells by electroporation, to achieve highly efficient transfection of primary alveolar epithelial type II (ATII) cells. To quantify the amount of ATII cells effectively transfected, we applied a plasmid expressing GFP and assessed the amount of GFP-expressing cells by flow cytometry. Analysis of the nucleofected ATII cells revealed a concentration-dependent transfection efficiency of up to 50% when using 3-8 mu g plasmid DNA without affecting cell viability. Nucleofection of cultured A549 and H441 cells yielded similar transfection rates. Importantly, nucleofection of ATII cells did not interfere with the integrity of ATII monolayers even with use of relatively high concentrations of plasmid DNA. In subsequent studies, we also efficiently delivered small interfering RNAs to ATII cells by nucleofection, thereby silencing Akt and the multiligand receptor megalin, which has been recently shown to play a key role in removal of excess protein from the alveolar space, and effectively inhibited megalin-driven uptake and transcellular transport of albumin in ATII cells. Thus we report successful transfection of primary rat alveolar epithelial cells with both plasmids and oligonucleotides via nucleofection with high viability and consistently good transfection rates without impairing key physiological properties of the cells.