van Leeuwen E B, van der Veen A Y, Hoekstra D, Engberts J B, Halie M R, van der Meer J, Ruiters M H
Department of Haematology, University of Groningen, The Netherlands.
Eur J Vasc Endovasc Surg. 1999 Jan;17(1):9-14. doi: 10.1053/ejvs.1998.0677.
This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells.
Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 10:1 ratio for concentrations of SAINT-2pp/DOPE: plasmid. Using these conditions, cell concentrations were lowered step-wise and we were able to transfect as few as one thousand cells with both methods. For detection of transfection of a small number of cells a sensitive assay was needed (Luciferase). A plasmid containing the neomycin resistance gene was used to determine the transfection rate expressed in colony forming units by counting colonies after selection. At low plasmid concentrations this transfection rate was within the same range for both electroporation and SAINT-2pp/DOPE transfection. Fluorescent in situ hybridisation of metaphase chromosomes of transfected endothelial cells using the plasmid as a probe showed that stable integration was possible with both methods.
Electroporation and a synthetic amphiphile, SAINT-2pp, provide the possibility of transfecting small numbers of cells resulting in stable integration of low plasmid concentrations. The availability of this technology is important in order to obtain functional endothelial cell lines from various human blood vessels for research purposes.
本研究比较了电穿孔法与合成两亲分子（SAINT-2pp/DOPE）转染少量人内皮细胞的效率。
对最佳转染条件进行了测试，电穿孔法的最佳条件似乎为400V和960微法，SAINT-2pp/DOPE与质粒的浓度比为10:1。使用这些条件，逐步降低细胞浓度，两种方法都能够转染低至一千个细胞。为了检测少量细胞的转染情况，需要一种灵敏的检测方法（荧光素酶）。使用含有新霉素抗性基因的质粒，通过在选择后计数菌落来确定以集落形成单位表示的转染率。在低质粒浓度下，电穿孔法和SAINT-2pp/DOPE转染法的转染率处于同一范围内。使用该质粒作为探针，对转染内皮细胞的中期染色体进行荧光原位杂交，结果表明两种方法都有可能实现稳定整合。
电穿孔法和合成两亲分子SAINT-2pp提供了转染少量细胞并使低浓度质粒稳定整合的可能性。为了从各种人体血管获得用于研究目的的功能性内皮细胞系，这项技术的可用性很重要。
